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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a global health threat with the potential to cause severe disease manifestations in the lungs. Although COVID-19 has been extensively characterized clinically, the factors distinguishing SARS-CoV-2 from other respiratory viruses are unknown. Here, we compared the clinical, histopathological, and immunological characteristics of patients with COVID-19 and pandemic influenza A(H1N1). We observed a higher frequency of respiratory symptoms, increased tissue injury markers, and a histological pattern of alveolar pneumonia in pandemic influenza A(H1N1) patients. Conversely, dry cough, gastrointestinal symptoms and interstitial lung pathology were observed in COVID-19 cases. Pandemic influenza A(H1N1) was characterized by higher levels of IL-1RA, TNF-α, CCL3, G-CSF, APRIL, sTNF-R1, sTNF-R2, sCD30, and sCD163. Meanwhile, COVID-19 displayed an immune profile distinguished by increased Th1 (IL-12, IFN-γ) and Th2 (IL-4, IL-5, IL-10, IL-13) cytokine levels, along with IL-1β, IL-6, CCL11, VEGF, TWEAK, TSLP, MMP-1, and MMP-3. Our data suggest that SARS-CoV-2 induces a dysbalanced polyfunctional inflammatory response that is different from the immune response against pandemic influenza A(H1N1). Furthermore, we demonstrated the diagnostic potential of some clinical and immune factors to differentiate both diseases. These findings might be relevant for the ongoing and future influenza seasons in the Northern Hemisphere, which are historically unique due to their convergence with the COVID-19 pandemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer (MC) has declared a shared affiliation, with no collaboration with the author (SK), to the handling editor, at the time of review., (Copyright © 2021 Choreño-Parra, Jiménez-Álvarez, Cruz-Lagunas, Rodríguez-Reyna, Ramírez-Martínez, Sandoval-Vega, Hernández-García, Choreño-Parra, Balderas-Martínez, Martinez-Sánchez, Márquez-García, Sciutto, Moreno-Rodríguez, Barreto-Rodríguez, Vázquez-Rojas, Centeno-Sáenz, Alvarado-Peña, Salinas-Lara, Sánchez-Garibay, Galeana-Cadena, Hernández, Mendoza-Milla, Domínguez, Granados, Mena-Hernández, Pérez-Buenfil, Domínguez-Cheritt, Cabello-Gutiérrez, Luna-Rivero, Salas-Hernández, Santillán-Doherty, Regalado, Hernández-Martínez, Orozco, Ávila-Moreno, García-Latorre, Hernández-Cárdenas, Khader, Zlotnik and Zúñiga.)

Background: Periodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS)., Methods: Human PDLCs were incubated with baicalein (0-100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot., Results: MTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs., Conclusions: With the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Endoplasmic reticulum (ER) stress associated proteins contribute to the pathogenesis of rheumatoid arthritis (RA) through affecting synoviocyte proliferation and proinflammatory cytokine production. The role of DERL3, an ER-associated degradation component, in joint inflammation of RA was explored. Synovial tissues from RA and osteoarthritis (OA) patients were collected, and in RA synovial tissue, DERL3 showed up-regulation and significantly positive correlation with the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-1. Immunofluorescence result suggested DERL3 was located in fibroblast-like synoviocytes (FLS). Among different inflammatory stimuli, DERL3 could be up-regulated by TNF-α stimulation in FLS. Under TNF-α stimulation, knocking down DERL3, the expression of IL-6, IL-8, MMP-1, MMP-13 was reduced and the activation of nuclear factor kappa B (NF-κB) signaling pathway was inhibited. In pristane-induced arthritis (PIA) rat model, Derl3 was up-regulated in synovial tissue and disease was attenuated after intraarticular injection of siDerl3. Overall, we conclude that TNF-α inducing DERL3 expression promotes the inflammation of FLS through activation of NF-κB signaling pathway, suggesting DERL3 plays important roles in the pathogenesis of RA and is a promising therapeutic target., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Iguratimod, a novel disease-modifying anti-rheumatic drug for the treatment of rheumatoid arthritis, has been approved in China and Japan. Here, we aimed to find whether iguratimod can inhibit the aggressive behavior and promote apoptosis of rheumatoid fibroblast-like synoviocytes (RA-FLSs)., Methods: The proliferation of RA-FLSs was assessed by 5-ethynyl-2'-deoxyuridine test and Cell Counting Kit-8. Migration and invasion were determined by the wound test and a transwell assay. Apoptosis was tested by flow cytometry. The mRNA expression of matrix metalloproteinases (MMPs) and proinflammatory cytokines in RA-FLSs were measured by quantitative PCR and ELISA. To gain insight into the molecular signaling mechanisms, we determined the effect of iguratimod on the activation of mitogen-activated protein kinases (MAPK) signaling pathways by the cellular thermal shift assay (CETSA) and western blot., Results: Iguratimod treatment significantly reduced the proliferation, migration, and invasive capacities of RA-FLSs in a dose-dependent manner in vitro . MMP-1, MMP-3, MMP-9, Interleukin-6 (IL-6), and monocyte chemoattractant protein-1 mRNA and protein levels were all decreased after treatment with iguratimod. Furthermore, tumor necrosis factor-alpha- (TNF- α -) induced expression of phosphorylated c-Jun N-terminal kinases (JNK) and P38 MAPK were inhibited by iguratimod. Additionally, iguratimod promoted the apoptosis of RA-FLSs. Most importantly, iguratimod was shown to directly interact with JNK and P38 protein by CETSA assay. Moreover, activating transcription factor 2 (ATF-2), a substrate of both JNK and P38, was suppressed by iguratimod., Conclusions: Our findings suggested that the therapeutic effects of iguratimod on RA might be, in part, due to targeting the aggressive behavior and apoptosis of RA-FLSs., Competing Interests: The authors confirm that this article content has no conflicts of interest., (Copyright © 2019 Jin Lin et al.)

Reproductive tract pathology caused by Chlamydia trachomatis infection is an important global cause of human infertility. To better understand the mechanisms associated with Chlamydia -induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to Chlamydia Disruption of TLR3 function in these cells significantly diminished the Chlamydia -induced synthesis of several inflammation biomarkers, including interferon beta (IFN-β), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rβ, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the Chlamydia -induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that Chlamydia infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within Chlamydia inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection., (Copyright © 2019 American Society for Microbiology.)

Skin is the outer tissue layer and is a barrier protecting the body from various external stresses. The fresh water green edible algae Prasiola japonica has antiviral, antimicrobial, and anti-inflammatory properties; however, few studies of its effects on skin-protection have been reported. In this study, Prasiola japonica ethanol extract (Pj-EE) was prepared, and its skin-protective properties were investigated in skin keratinocytes. Pj-EE inhibited ROS production in UVB-irradiated HaCaT cells without cytotoxicity. Pj-EE also suppressed the apoptotic death of UVB-irradiated HaCaT cells by decreasing the generation of apoptotic bodies and the proteolytic activation of apoptosis caspase-3, -8, and -9. Moreover, Pj-EE downregulated the mRNA expression of the inflammatory gene cyclooxygenase-2 (COX-2), the pro-inflammatory cytokine genes interleukin (IL)-1 β , IL-8, IL-6, tumor necrosis factor (TNF)- α , and interferon (IFN)- γ , and the tissue remodeling genes matrix metalloproteinase (MMP)-1, -2, -3, and -9. The Pj-EE-induced anti-inflammatory effect was mediated by suppressing the activation of nuclear factor-kappa B (NF- κ B) signaling pathway in the UVB-irradiated HaCaT cells. Taken together, these results suggest that Pj-EE exerts skin-protective effects through anti-oxidant, anti-apoptotic, and anti-inflammatory activities in skin keratinocytes.

Background/aims: Osteoarthritis (OA) is a multifactorial disease that is associated with inflammation in joints. The purpose of the present study was to investigate the anti-inflammatory activity and mechanism of morin on human osteoarthritis chondrocytes stimulated by IL-1β., Methods: The levels of NO and PGE2 were measured by the Griess method and ELISA. The levels of MMP1, MMP3, and MMP13 were also measured by ELISA., Results: The results revealed that IL-1β significantly increased the production of NO, PGE2, MMP1, MMP3, and MMP13. Additionally, the increases were significantly attenuated by treatment with morin. Furthermore, IL-1β-induced NF-κB activation was suppressed by morin. In addition, the expression of Nrf2 and HO-1 were increased by morin and knockdown of Nrf2 could prevent the anti-inflammatory effects of morin., Conclusion: In conclusion, this study suggested that morin attenuated IL-1β-induced inflammation by activating the Nrf2 signaling pathway., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

We previously isolated Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) from tobacco leaves. The NMMP1 gene encodes a highly conserved, Zn-containing catalytic protease domain that functions as a factor in the plant's defense against bacterial pathogens. Expression of NMMP1 was strongly induced during interactions between tobacco and one of its pathogens, Phytophthora infestans. To elucidate the role of the NMMP1 in defense of N. benthamiana against fungal pathogens, we performed gain-of-function and loss-of-function studies. NMMP1-overexpressing plants had stronger resistance responses against P. infestans infections than control plants, while silencing of NMMP1 resulted in greater susceptibility of the plants to the pathogen. This greater susceptibility correlated with fewer NMMP1 transcripts than the non-silenced control. We also examined cell death as a measure of disease. The amount of cell death induced by the necrosis-inducing P. infestans protein 1, PiNPP1, was dependent on NMMP1 in N. benthamiana. Potato plants overexpressing NMMP1 also had enhanced disease resistance against P. infestans. RT-PCR analysis of these transgenic potato plants revealed constitutive up-regulation of the potato defense gene NbPR5. NMMP1-overexpressing potato plants were taller and produced heavier tubers than control plants. We suggest a role for NMMP1in pathogen defense and development., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.

In the USA, increased cerebrospinal fluid (CSF) inflammatory cytokines have been observed in antiretroviral therapy (ART)-naive, HIV-seropositive individuals with HIV-associated neurocognitive disorder (HAND). We characterized the relationship between HAND and CSF biomarker expression in ART-naive, HIV-seropositive individuals in Rakai, Uganda. We analyzed CSF of 78 HIV-seropositive, ART-naive Ugandan adults for 17 cytokines and 20 neurodegenerative biomarkers via Luminex multiplex assay. These adults underwent neurocognitive assessment to determine their degree of HAND. We compared biomarker concentrations between high and low CD4 groups and across HAND classifications, adjusting for multiple comparisons. Individuals with CD4 <200 cells/μL (N = 38) had elevated levels of CSF Interleukin (IL)-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-α, matrix metalloproteinase (MMP)-1, MMP-7, and S100 calcium-binding protein B (S100B) and lower levels of amyloid β42. Individuals with CD4 351-500 cells/μL (N = 40) had significantly higher CSF levels of interleukin (IL)-1β, amyloid β42, and soluble receptor for advanced glycation end products (sRAGE). Increasing levels of S100B, platelet-derived growth factor-AA (PDGF-AA), brain-derived neurotrophic factor (BDNF), and sRAGE were associated with decreased odds of mild neurocognitive disorder (n = 22) or HIV-associated dementia (n = 15) compared with normal function (n = 30) or asymptomatic neurocognitive impairment (n = 11). Increased levels of interferon (IFN)-γ were associated with increased odds of mild neurocognitive impairment or HIV-associated dementia relative to normal or asymptomatic neurocognitive impairment. Proinflammatory CSF cytokines, chemokines, and neurodegenerative biomarkers were present in increasing concentrations with advanced immunosuppression and may play a role in the development of HAND. The presence of select CNS biomarkers may also play a protective role in the development of HAND.

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study aimed to investigate the therapeutic mechanisms of nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids in osteoarthritis (OA). The CHON‑002 human chondrocyte cell line was used in the study. The levels of the cytokines, interleukin (IL)‑1β, IL‑6, IL‑8 and IL‑10, released by cells treated with tumor necrosis factor‑α (TNF‑α) were determined by ELISA. Levels of collagen I, aggrecan, matrix metalloproteinase (MMP)‑1, MMP‑13, signal transducer and activator of transcription (STAT) 3, nuclear factor‑κB (NF‑κB) subunit p65 and inhibitory subunit of NF‑κB (IκB) following treatment with IL‑1β, IL‑6, IL‑8 or IL‑10 were assessed by western blot. Levels of IL‑6 and IL‑8 were measured by ELISA following administration of TNF‑α combined with certain drugs. In addition, these parameters were evaluated by western blot following incubation with drugs in combination with IL‑6 or IL‑8 and after knockdown of STAT3, by addition of small interfering RNA (siRNA)‑STAT3 (siSTAT3), an inhibitor of the proteasome (MG132) or both. IL‑1β, IL‑6, IL‑8 and IL-10 were upregulated by TNF‑α. Addition of IL‑6 or IL‑8 led to increased collagen I, MMP‑1 and MMP‑13 protein levels, and also promoted STAT3 phosphorylation and increased the expression of NF‑κB subunit p65, but had no effect on aggrecan protein levels. When siSTAT3 and MG132 treatment was combined, levels of collagen I, MMP‑1 and MMP‑13 were reduced. Additionally, levels of IL‑6 and IL‑8 were significantly decreased by prednisone, ibuprofen and betamethasone. However, no significant differences were observed following treatment with piroxicam or indomethacin. In combination with IL‑6 or IL‑8, prednisone, ibuprofen and betamethasone significantly reduced the levels of collagen I, MMP‑1 and MMP‑13, and inactivated NF‑κB and STAT3 pathways. In conclusion, prednisone, ibuprofen and betamethasone may prevent OA by suppressing the expression of IL‑6 and IL‑8, subsequently inactivating NF‑κB and STAT3 pathways, and ultimately, leading to decreased levels of collagen I, MMP‑1, and MMP‑13.

The accumulation and infiltration of mast cells are found in osteoarthritic lesions in humans and rodents. Nonetheless, the roles of mast cells in osteoarthritis are almost unknown. Although Viscum coloratum has various beneficial actions, its effect on allergic and osteoarthritic responses is unknown. In this study, we established an in vitro model of mast cell-mediated osteoarthritis and investigated the effect of the ethanol extract of Viscum coloratum (VEE) on IgE/antigen (IgE/Ag)-activated mast cells and mast cell-derived inflammatory mediator (MDIM)-stimulated chondrocytes. The anti-allergic effect of VEE was evaluated by degranulation, inflammatory mediators, and the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. The anti-osteoarthritic action of VEE was evaluated by cell migration, and the expression, secretion, and activity of MMPs in MDIM-stimulated SW1353 cells. VEE significantly inhibited degranulation (IC 50 : 93.04 μg/mL), the production of IL-4 (IC 50 : 73.28 μg/mL), TNF-α (IC 50 : 50.59 μg/mL), PGD₂ and LTC₄, and activation of the FcεRI signaling cascade in IgE/Ag-activated RBL-2H3 cells. Moreover, VEE not only reduced cell migration but also inhibited the expression, secretion, and/or activity of MMP-1, MMP-3, or MMP-13 in MDIM-stimulated SW1353 cells. In conclusion, VEE possesses both anti-allergic and anti-osteoarthritic properties. Therefore, VEE could possibly be considered a new herbal drug for anti-allergic and anti-osteoarthritic therapy. Moreover, the in vitro model may be useful for the development of anti-osteoarthritic drugs.

Psoriasis is a debilitating chronic inflammatory disease. In addition to the characteristic effects on the skin, chronic inflammation associated with the disease is recognized to contribute to cardiovascular, hepatic and renal comorbidities. Immature myeloid regulatory cells, known as myeloid‑derived suppressor cells (MDSCs), have been demonstrated to accumulate in various diseases and chronic inflammatory states, including inflammatory bowel disease and various types of cancer. The results of the present study, obtained using flow cytometry and cell culture analysis of peripheral blood mononuclear cells from psoriasis and healthy patients, revealed that MDSC levels are significantly increased in the blood of patients with psoriasis compared with healthy controls. Furthermore, these cells are capable of producing various molecules, including matrix metalloproteinase‑9 and‑1, interleukin‑8, growth‑related oncogene, and monocyte chemoattractant protein 1. These molecules may recruit additional immune cells involved in the pathogenesis of the disease, and contribute to the chronic inflammatory state in these patients. Therefore, MDSCs, which have various immune regulatory functions, may contribute to the pathogenesis of psoriasis as a systemic inflammatory disease.

Objective: Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs., Methods: MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6., Results: The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs., Conclusion: Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs., (© 2016, American College of Rheumatology.)

To inhibit the immune inflammation in the allografts can be beneficial to organ transplantation. This study aims to induce the donor antigen specific regulatory T cells (Treg cell) inhibit the immune inflammation in the allograft heart. In this study, peripheral exosomes were purified from the mouse serum. A heart transplantation mouse model was developed. The immune inflammation of the allograft heart was assessed by histology and flow cytometry. The results showed that the donor antigen-specific T helper (Th)2 pattern inflammation was observed in the allograft hearts; the inflammation was inhibited by immunizing the recipient mice with the donor-derived exosomes. Purified peripheral exosomes contained integrin MMP1a; the latter induced CD4(+) T cells to express Fork head protein-3 and transforming growth factor (TGF)-β via inhibiting the Th2 transcription factor, GATA binding protein 3, in CD4(+) T cells. Administration with the donor-derived exosomes significantly prolonged the allograft heart survival. We conclude that the donor-derived peripheral exosomes have the capacity to inhibit the immune inflammation in the allograft heart via inducing specific Treg cells, implicating that administration with the donor-derived exosomes may be beneficial to cardiac transplantation.

We have examined peripheral blood neutrophils from 123 patients with primary endometrial cancer at stage Ia. Receptor system and the ability of neutrophils to form extracellular traps were assessed by fluorescence microscopy, the spontaneous production of cytokines IL-2, IFN-γ, g-CSF, matrix metalloproteinases-1,9,13 by the method of enzyme-linked immunosorbent assay, phagocytic activity, myeloperoxidase activity, the level of cationic proteis activity in NBT-test were evaluated by cytochemical methods, activity of neutrophils in the spontaneous NBT-test was used to evaluate the oxygen-dependent bactericidal action of neutrophils. The topology and the rigidity of the membrane of neutrophils were assessed by scanning probe microscopy. We have shown that the increase in the relative number of neutrophils lead to a change in their receptor system, aerobic and anaerobic cytotoxicity and ability to phagocytosis are enchanced while reducing NET-activity. We have observed a change in the secretory activity of neutrophils, which is characterized by increased level of MMP-1, possibly initiated by enhanced production of reactive oxygen species, by a reduction in the IL-2 level (inductor of cytotoxic activity) and a sharp increase in the level of the G-CSF. Architectonics of neutrophils in the case of endonetrial cancer at stage Ia is characterized by changing the shape and loss of grit. The rigidity of the cell membrane decreased. Changes in the morphology of neutrophils on the background of the continuing hyperactivity suggests that a state of balance between the immune system and the tumor is already in stage Ia endometrial cancer.

Background: Tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) are elevated in pleural fluids of tuberculous pleuritis (TBP) where pleural mesothelial cells (PMCs) conduct the first-line defense against Mycobacterium tuberculosis (MTB). However, the clinical implication of TNF-α and MMPs in TBP and the response of PMCs to MTB infection remain unclear., Methods: We measured pleural fluid levels of TNF-α and MMPs in patients with TBP (n = 18) or heart failure (n = 18) as controls. Radiological scores for initial effusion amount and residual pleural fibrosis at 6-month follow-up were assessed. In vitro human PMC experiments were performed to assess the effect of heat-killed M. tuberculosis H37Ra (MTBRa) on the expression of TNF-α and MMPs., Results: As compared with controls, the effusion levels of TNF-α, MMP-1 and MMP-9 were significantly higher and correlated positively with initial effusion amount in patients with TBP, while TNF-α and MMP-1, but not MMP-9, were positively associated with residual pleural fibrosis of TBP. Moreover, effusion levels of TNF-α had positive correlation with those of MMP-1 and MMP-9 in TBP. In cultured PMCs, MTBRa enhanced TLR2 and TLR4 expression, activated ERK signaling, and upregulated TNF-α mRNA and protein expression. Furthermore, knockdown of TLR2, but not TLR4, significantly inhibited ERK phosphorylation and TNF-α expression. Additionally, both MTBRa and TNF-α markedly induced MMP-1 and MMP-9 synthesis in human PMCs, and TNF-α neutralization substantially reduced the production of MMP-1, but not MMP-9, in response to MTBRa stimulation., Conclusion: MTBRa activates TLR2/ERK signalings to induce TNF-α and elicit MMP-1 and MMP-9 in human PMCs, which are associated with effusion volume and pleural fibrosis and may contribute to pathogenesis of TBP. Further investigation of manipulation of TNF-α and MMP expression in pleural mesothelium may provide new insights into the mechanisms and rational treatment strategies for TBP.

Background: Atopic dermatitis (AD) is the most common inflammatory disease. The prevalence of allergic contact dermatitis to allergens (eg, fragrance) is higher in patients with AD, despite a trend toward weaker clinical allergic contact dermatitis reactions. The role of the AD skin phenotype in modulating allergic sensitization to common sensitizers has not been evaluated., Objective: We sought to investigate whether patients with AD have altered tissue immune responses on allergen challenge., Methods: Gene expression and immunohistochemistry studies were performed on biopsy specimens from 10 patients with AD and 14 patients without AD patch tested with common contact allergens (nickel, fragrance, and rubber)., Results: Although 1085 differentially expressed genes (DEGs) were commonly modulated in patch-tested skin from patients with AD and patients without AD versus control skin, 1185 DEGs were uniquely altered in skin from patients without AD, and only 246 DEGs were altered in skin from patients with AD. Although many inflammatory products (ie, matrix metalloproteinase 12/matrix metalloproteinase 1/S100A9) were upregulated in both groups, higher-magnitude changes and upregulation of interferon responses were evident only in the non-AD group. Stratification by allergen showed decreased expression of immune, TH1-subset, and TH2-subset genes in nickel-related AD responses, with increased TH17/IL-23 skewing. Rubber/fragrance showed similar trends of lesser magnitude. Negative regulators showed higher expression in patients with AD., Conclusions: Through contact sensitization, our study offers new insights into AD. Allergic immune reactions were globally attenuated and differentially polarized in patients with AD, with significant decreases in levels of TH1 products, some increases in levels of TH17 products, and inconsistent upregulation in levels of TH2 products. The overall hyporesponsiveness in skin from patients with background AD might be explained by baseline immune abnormalities, such as increased TH2, TH17, and negative regulator levels compared with those seen in non-AD skin., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Objectives: Due to their role in inflammatory metabolic diseases, we hypothesised that free fatty acids (FFA) are also involved in inflammatory joint diseases. To test this hypothesis, we analysed the effect of FFA on synovial fibroblasts (SF), human chondrocytes and endothelial cells. We also investigated whether the toll-like receptor 4 (TLR4), which can contribute to driving arthritis, is involved in FFA signalling., Methods: Rheumatoid arthritis SF, osteoarthritis SF, psoriatic arthritis SF, human chondrocytes and endothelial cells were stimulated in vitro with different FFA. Immunoassays were used to quantify FFA-induced protein secretion. TLR4 signalling was inhibited extracellularly and intracellularly. Fatty acid translocase (CD36), responsible for transporting long-chain FFA into the cell, was also inhibited., Results: In rheumatoid arthritis synovial fibroblasts (RASF), FFA dose-dependently enhanced the secretion of the proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, as well as the matrix-degrading enzymes pro-MMP1 and MMP3. The intensity of the response was mainly dependent on the patient rather than on the type of disease. Both saturated and unsaturated FFA showed similar effects on RASF, while responses to the different FFA varied for human chondrocytes and endothelial cells. Extracellular and intracellular TLR4 inhibition as well as fatty acid transport inhibition blocked the palmitic acid-induced IL-6 secretion of RASF., Conclusions: The data show that FFA are not only metabolic substrates but may also directly contribute to articular inflammation and degradation in inflammatory joint diseases. Moreover, the data suggest that, in RASF, FFA exert their effects via TLR4 and require extracellular and intracellular access to the TLR4 receptor complex., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objectives: Nickel (Ni) is one of the main metal elements in orthodontic and prosthetic devices. Different effects of Ni are described ranging from an induction of local inflammation to allergy and cancerous/mutagenic properties. Inflammatory reactions are frequently observed in the oral cavity, but the interrelationship of Ni with those events is still unknown. Therefore, we focused on the impact of Ni on inflammation in vitro., Methods: In accordance to previous immersion tests of our lab, human gingival fibroblasts (HGFs) (n=6) were exposed to a pro-inflammatory environment using interleukin-1 beta (IL-1β) and additionally stimulated with different Ni(II) concentrations (400 and 4000ng/ml). At varying time points the expression of pro- and anti-inflammatory as well as matrix degeneration proteins, i.e. MMPs, were analyzed. Furthermore, proliferation assays, wound healing tests and the detection of NF-κB activation were conducted. Unstimulated HGFs served as control., Results: Our experiments showed that low clinical average Ni(II) levels did not alter pro-inflammatory cytokines significantly compared to control (p>0.05). Instead, a 10-fold higher dose up-regulated these mediators significantly in a time-dependent manner (p<0.01). This was even more pronounced combining both Ni(II) concentrations with an inflammatory condition (p<0.001), MMP expressions were in line with our findings (p<0.001). The mRNA data were supported by proliferation and wound closure assays (p<0.001). However, the combination of both stimuli induced contradictory results. Analyzing NF-κB activation revealed that our results may be in part attributed to NF-κB., Significance: Our in vitro study implicated that Ni(II) has various modifying effects on IL-1β-induced inflammatory processes depending on the concentration., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Degradation of collagen type II caused by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) is one of the major pathological characteristics of osteoarthritis (OA). Dimethyl fumarate (DMF) is a medication approved by the US Food and Drug Administration (FDA) as an oral multiple sclerosis (MS) therapy. In this study, we found that DMF ameliorated collagen type II degradation by inhibiting the expression of MMP-1, MMP-3, and MMP-13 caused by TNF-α. Mechanistically, DMF attenuated MMPs expression by suppressing JAK/STAT3 pathway. These findings imply that DMF treatment might be a potential therapeutic strategy for chondroprotective therapy.

Purpose: Primary cutaneous T-cell lymphomas (CTCL) are neoplastic disorders of skin-homing T cells. Affected skin areas show morphologic similarities with alterations in other T-cell-mediated dermatoses. Furthermore, as in atopic dermatitis but in contrast with psoriasis, patients with CTCL are frequently afflicted by cutaneous bacterial infections that support the survival of lymphoma cells. Our aim was to investigate the mechanisms of elevated susceptibility to cutaneous infections in patients with CTCL., Experimental Design: Skin samples from CTCL, psoriasis, and atopic dermatitis patients were used to illuminate the antibacterial competence status and the presence of its modulating cytokines. For substantiation of findings, 3-dimensional epidermis models, isolated and in vitro generated Th-subpopulations, were applied. Parameters were analyzed via qPCR and IHC., Results: CTCL lesions compared with psoriatic lesions presented an impaired upregulation of antibacterial proteins (ABPs), with levels even below those in atopic dermatitis. This was associated with a relative deficiency of the ABP-inducing cytokine IL-17 and a strong presence of the ABP-downregulating cytokine IL-13. The simultaneous presence of the Th17-cell cytokine IL-26 indicated that IL-17 deficiency in CTCL lesions results from functional deviation of Th17 cells. Accordingly, IL-17 but not IL-26 production by Th17 cells in vitro was inhibited by IL-4Rα ligand. Levels of other ABP inducers were comparable between CTCL and psoriasis lesions. The same was true about IL-22/TNF-α targets, including the keratinocyte hyper-regeneration marker K16 and the matrix-degrading enzyme MMP1., Conclusion: Our results suggest that the cutaneous bacterial infections in CTCL are caused by impaired ABP induction as consequence of Th2-mediated biased Th17-cell function., (©2014 American Association for Cancer Research.)

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The potential therapeutic effects of Costa Rican guava (Psidium friedrichsthalianum) extracts for chronic obstructive pulmonary disease were examined. The ethyl acetate fraction displayed the highest antioxidant activity, as compared to the hexane, chloroform, and n-butanol fractions, as well as the crude extract. This fraction was evaluated for its anti-inflammatory activity response relationship against interleukin-8 (IL-8) and inhibition of matrix metalloproteinase-1 (MMP-1) expression before and after treatment with cigarette smoke. The ethyl acetate fraction exhibited inhibitory activity against IL-8 production and MMP-1 expression, showing the most potent inhibitory activities in both assays at 100μg/mL, and nine compounds (1-9) were found. Phenolic compounds 1-O-trans-cinnamoyl-β-d-glucopyranose (2), ellagic acid (3), myricetin (4), quercitrin (7), and quercetin (9) were identified using standard compounds or literature reports from related species. Compounds 1, 5, 6, and 8 were tentatively identified as 1,5-dimethyl citrate (1), sinapic aldehyde 4-O-β-d-glucopyranose (5), 3,3',4-tri-O-methylellagic acid-4'-O-d-glucopyranoside (6), and 1,3-O-diferuloylglycerol (8), All nine compounds are reported for the first time in Costa Rican guava., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aggressive phenotype of RA synovial fibroblasts (RASF) is characterised by the increased expression of matrix metalloproteinase (MMP)-1 as well as the small ubiquitin like modifier (SUMO)-1 and decreased expression of SUMO-specific protease SENP1. Since we showed an increased activity of acetyltransferases in this autoimmune disease, we wanted to analyze whether this affects the expression of MMP-1 and can be reversed by the reconstitution of SENP1. In RASF, the acetylation of histone H4 was significantly increased in the distal region of the MMP-1 promoter by 274 +/- 36% compared to OASF. Most interestingly, overexpression of SENP1 in RASF decreased acetylation specifically in this region by 51 +/- 0.5% and globally by 73 +/- 11%. Furthermore, the overexpression of SENP1 resulted in a downregulation of MMP-1 at both the mRNA (58 +/- 7%) and protein levels (28 +/- 6%), significantly reduced the invasiveness of RASF (from 34 +/- 9% to 2 +/- 2%) and led to an accumulation of histone deacetylase 4 (HDAC4) on the MMP-1 promoter (197 +/- 36%). Interestingly, SENP1 failed to modulate the expression of MMP-1 in the cells silenced for HDAC4. This is the first study linking the SUMOylation pathway and the production of MMP-1 to an epigenetic control mechanism mediated through histone acetylation which has a functional consequence for the invasiveness of RASF.

Purpose: Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs., Methods: We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3 h, 24 h, 48 h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies., Results: All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48 h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site., Conclusion: Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of disease.

Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.

Even in the absence of an adaptive immune system in murine models, lymphatic dilatation and dysfunction occur in filarial infections, although severe irreversible lymphedema and elephantiasis appears to require an intact adaptive immune response in human infections. To address how filarial parasites and their antigens influence the lymphatics directly, human lymphatic endothelial cells were exposed to filarial antigens, live parasites, or infected patient serum. Live filarial parasites or filarial antigens induced both significant LEC proliferation and differentiation into tube-like structures in vitro. Moreover, serum from patently infected (microfilaria positive) patients and those with longstanding chronic lymphatic obstruction induced significantly increased LEC proliferation compared to sera from uninfected individuals. Differentiation of LEC into tube-like networks was found to be associated with significantly increased levels of matrix metalloproteases and inhibition of their TIMP inhibitors (Tissue inhibitors of matrix metalloproteases). Comparison of global gene expression induced by live parasites in LEC to parasite-unexposed LEC demonstrated that filarial parasites altered the expression of those genes involved in cellular organization and development as well as those associated with junction adherence pathways that in turn decreased trans-endothelial transport as assessed by FITC-Dextran. The data suggest that filarial parasites directly induce lymphangiogenesis and lymphatic differentiation and provide insight into the mechanisms underlying the pathology seen in lymphatic filariasis.

Background: Acute irritant-induced asthma (IrIa) or reactive airways dysfunction syndrome is caused by exposure to a high concentration of an agent. The long-term pathologic consequences of IrIa remain thus far unknown., Objective: The aim of our study was to investigate the chronic airway inflammation and remodeling that occur in association with IrIa., Methods: Ten subjects with a history of IrIa (mean interval of 10.9 years, minimum of 4 years, since the inhalational accident) underwent bronchoscopy followed by bronchoalveolar lavage and bronchial biopsies. Immunologic and morphologic data from patients with IrIa were compared with those of patients with mild to moderate asthma as well as healthy controls., Results: Bronchoalveolar lavage fluid analysis showed increased eosinophil and neutrophil counts in 30% and 60% of subjects with IrIa, respectively. In the supernatant of bronchoalveolar lavage, we found a significant increase in the majority of mediators compared with healthy subjects and a significant increase in eosinophilic cationic protein, IL-8, basic fibroblast growth factor, and matrix metalloproteinase 1 compared with control patients with asthma. Evaluation of basement membrane thickness (subepithelial fibrosis) demonstrated a significant increase in patients with IrIa compared with healthy subjects and subjects with asthma. Basement membrane thickness also significantly correlated with the PC(20) value. The epithelial cell detachment showed an elevated although not significant trend compared with subjects with asthma and control subjects. Immunocytochemical analysis demonstrated increases in the number of eosinophil cationic protein and TGF-beta1-positive cells compared with healthy controls., Conclusion: This study provides evidence of a significant eosinophilic and neutrophilic inflammation as well as remodeling in IrIa many years after an inhalational accident.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate <30 ml/min/1.73 m(2) in univariate analyses. MMP-1 staining, but not TIMP-1 staining, was inversely correlated with increased glomerular fibrosis (r = -0.40). In multivariable analysis, diabetes was robustly associated with decreased staining intensity. This study indicates that in human subjects, the long-term sequelae of diseases such as diabetes that cause chronic renal failure result in decreased TIMP-1 and MMP-1 proteins in renal glomeruli. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Introduction: The role of adiponectin in the pathogenesis of arthritis is still controversial. This study was performed to examine whether adiponectin is involved in joint inflammation and destruction in rheumatoid arthritis (RA) in relation to the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs)., Methods: Synovial cells from RA patients were treated with adiponectin or interleukin (IL)-1beta for 24 hours. The culture supernatant was collected and analyzed for the levels of IL-6, IL-8, prostaglandin E2 (PGE2), VEGF, and MMPs by enzyme-linked immunosorbent assay. The levels of adiponectin, VEGF, MMP-1, and MMP-13 in the joint fluids from 30 RA or osteoarthritis (OA) patients were also measured., Results: Adiponectin at the concentration of 10 microg/mL stimulated the production of IL-6, IL-8, and PGE2 in RA fibroblast-like synoviocytes (FLSs), although the level of these was much lower than with 1 ng/mL IL-1beta. However, adiponectin stimulated the production of VEGF, MMP-1, and MMP-13 at the same level as IL-1beta. In addition, the level of adiponectin and MMP-1 in the joint fluid of RA patients was significantly higher than in OA patients. Adiponectin was positively correlated with VEGF in RA patients but not in OA patients, while the level of MMPs in joint fluid was not correlated with adiponectin in either RA or OA patients., Conclusions: Adiponectin may play a significant role in the pathogenesis of RA by stimulating the production of VEGF and MMPs in FLSs, leading to joint inflammation and destruction, respectively.

Background: Localized scleroderma (LSc) is characterized by cutaneous fibrosis and various autoantibodies., Objective: To determine the presence or levels of antibodies (Abs) against matrix metalloproteinase (MMP)-1 and their clinical relevance in LSc., Methods: Anti-MMP-1 Ab was examined by ELISA (Enzyme-Linked ImmunoSorbent Assay) and immunoblotting using human recombinant MMP-1. MMP-1 collagenase activity was determined using biotinylated collagen as substrate and the amount of cleaved biotinylated fragments of collagen by MMP-1 was measured by ELISA., Results: LSc patients exhibited significantly elevated IgG anti-MMP-1 Ab levels relative to normal controls at similar level of patients with systemic sclerosis (SSc). However, IgG anti-MMP-1 Ab levels were comparable among the 3 LSc subgroups: morphea, linear scleroderma, and generalized morphea. When absorbance values higher than the mean+2S.D. of normal controls were considered positive, IgG or IgM anti-MMP-1 Ab was found in 46% and 49% of total LSc patients and SSc patients, respectively. Anti-MMP-1 Ab was detected most frequently in morphea patients (60%), followed by linear scleroderma patients (47%) and then generalized morphea patients (25%). LSc patients positive for IgG anti-MMP-1 Ab had elevated levels of IgG anti-single-stranded DNA Ab, IgG anti-nucleosome Ab, and shorter disease duration relative to those negative. The presence of anti-MMP-1 Ab in LSc patients was confirmed by immunoblotting. IgG isolated from LSc patients' sera positive for IgG anti-MMP-1 Ab by ELISA inhibited MMP-1 collagenase activity., Conclusion: These results suggest that anti-MMP-1 autoantibody is a novel autoantibody in LSc.

C-reactive protein (CRP) and oxidized LDL (ox-LDL) are associated with inflammatory lesions, such as coronary artery disease, in which monocytes and matrix metalloproteinases (MMPs) may play a major role in the rupture of atherosclerotic plaques. Monocytes are recruited to inflammation sites by monocyte chemoattractant protein-1 (MCP-1), which may also participate in the activation of monocytes. The objective of this study was to compare the individual and combined effect of CRP and ox-LDL on human monocyte MMP-1 and the role of MCP-1 in this effect. Although CRP or ox-LDL failed to induce MMP-1 in control monocytes, these molecules enhanced MMP-1 production induced by tumor necrosis factor alpha (TNF-alpha) and granulocyte macrophage-colony stimulating factor (GM-CSF) with a synergistic increase in MMP-1 occurring in the presence of both mediators. Enhancement of MMP-1 by CRP and ox-LDL was attributable to a differential increase in MCP-1 and prostaglandin E2(PGE2). CRP, at physiological concentrations, induced high levels of MCP-1 and relatively low levels of PGE2, whereas ox-LDL caused a significant enhancement of PGE2 with little affect on MCP-1. Accordingly, CRP- and ox-LDL-induced MMP-1 production by monocytes was inhibited by anti-MCP-1 antibodies and indomethacin, respectively. Moreover, addition of exogenous MCP-1 or PGE2 enhanced MMP-1 production by TNF-alpha- and GM-CSF-stimulated monocytes. These results show that the combination of CRP and ox-LDL can cause a synergistic enhancement of the role of monocytes in inflammation, first, by increasing MCP-1, which attracts more monocytes and directly enhances MMP-1 production by activated monocytes, and second, by elevating PGE2 production, which also leads to higher levels of MMP-1.

A novel link between development and immunity in insects is introduced. Transiently enhanced expression of lysozyme, gallerimycin and the insect metalloproteinase inhibitor was discovered at the onset of metamorphosis of the greater wax moth, Galleria mellonella. Relative quantification of mRNAs encoding for these antimicrobial peptides using real-time PCR documents their induced expression during transformation of last instar larvae into prepupae and upon injection of either recombinant interstitial collagenase (MMP-1) or small-sized fragments of collagen type IV. The latter were also found to stimulate both nuclear import of c-Rel-proteins in the fat body, implicating activation of Toll or Imd-related signaling pathways, and subsequent synthesis of antimicrobial peptides. Obtained results implicate that degradation of collagen-IV by either microbial metalloproteinases associated with invading pathogens or endogenous matrix metalloproteinases contributing to degradation of extracellular matrix during metamorphosis stimulate innate immune responses.

Objective: Synovial inflammation in rheumatoid arthritis (RA) leads to pannus tissue invasion and destruction of cartilage/bone matrix by proteinases. Our intention was to analyze some of the key matrix metalloproteinases (MMPs) in pannus tissue overlying evolving cartilage erosions in RA., Methods: Frozen tissue samples of pannus and synovium from advanced RA and synovium from osteoarthritic patients were used for immunohistochemical, western blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of MMP-1, -3, -13 and -14. Synovial fibroblast cultures, stimulated with tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), were analyzed with enzyme-linked immunosorbent assays (ELISA) and quantitative RT-PCR., Results: MMP-3 was highly expressed in pannus tissue compared with significantly lower expression levels of MMP-1, -13 and -14. In fibroblast cultures IL-1beta was a potent stimulus for MMP-3, whereas TNF-alpha was more potent for MMP-1., Conclusion: This is the first study to demonstrate quantitatively in real time that MMP-3 mRNA expression is clearly higher in advanced RA pannus tissue compared to parallel RA or osteoarthritic synovium. MMP-3 mRNA levels were also clearly overexpressed in RA pannus compared to MMP-1, -13 and -14. Advanced RA has previously been found to overexpress IL-1beta. The high expression of MMP-3 in pannus and IL-1beta, mediated stimulation of MMP-3 suggest that MMP-3 plays a significant role in the progression of erosions through the proteoglycan-rich cartilage matrix.

Background: Matrix metalloproteinases (MMP) play an essential role in tissue remodelling and menstruation and appear to be regulated by cytokines such as interleukin-1alpha (IL-1alpha). In order to investigate their role in the pathogenesis of endometriosis, the aim of the present study was to compare the protein localization of matrix metalloproteinase-1 (MMP-1) and of its main stimulatory cytokine IL-1alpha in eutopic and dystopic endometrium of patients with endometriosis., Methods: MMP-1 and IL-1alpha protein localization was analysed retrospectively in paired paraffin-embedded tissue biopsies obtained simultaneously from the endometrial cavity and from endometrial lesions of 37 patients with peritoneal or ovarian endometriosis and in cycling endometria from 37 women without endometriosis. Protein localization was demonstrated by immunohistochemistry; antibody specificity was confirmed by western blot analysis., Results: MMP-1 and IL-1alpha protein staining in women suffering from endometriosis was significantly more pronounced in endometriotic lesions than in eutopic endometrium. This held true for both epithelial MMP-1 and IL-1alpha staining (P < 0.006 and P < 0.001), and for stromal MMP-1 and IL-1alpha staining (P < 0.001 and P < 0.001). Furthermore, stromal MMP-1 and IL-1alpha were significantly co-expressed in dystopic endometriotic tissue (P = 0.045). Endometrial MMP-1 and IL-1alpha protein expression pattern in eutopic endometrium from women suffering from endometriosis, however, did not differ significantly from the pattern seen in healthy women., Conclusions: The increased expression of both matrix-degrading MMP-1 and its major stimulatory cytokine IL-1alpha in endometriotic lesions and the selective co-expression in the stroma of endometriotic foci clearly suggests their involvement in the pathogenic mechanisms leading to local invasion and tissue destruction.

Background: Little information is available regarding the role of matrix metalloproteinases (MMPs) in thoracic aortic aneurysms and dissections. We applied tissue microarray analysis to determine MMP profiles in a large group of surgically resected thoracic aneurysms and dissections., Methods: Specimens from 47 patients undergoing a variety of surgical procedures for thoracic aneurysm (n = 30) and dissection (n = 17) were included. Expression of MMP-1, MMP-2, and MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were assessed by aortic tissue microarray immunostaining. Matrix metalloproteinase and TIMP expression in aortic tissue was compared with seven control aortic specimens, free of any vascular disease., Results: Expression of MMP-1 and MMP-9 was significantly increased in aneurysm and aortic dissection patients compared with control specimens (p < 0.05). Expression of TIMP-2 was significantly increased in the entire patient group, compared with control specimens (p < 0.05). Aortic dissection patients had higher MMP-2 and MMP-9 expression than aortic aneurysm patients in areas of disease. Compared with control patients, the MMP-9 to TIMP-1 ratio (a relative index of proteolytic state) was increased in both the aortic aneurysm and dissection groups (p < 0.05)., Conclusions: The increased MMP expression in aortic aneurysms and dissections indicates a metamorphosis in the aneurysm wall toward increased proteolysis compared with the normal aorta. Furthermore, we find even higher MMP-2 and MMP-9 presence in aortic dissection. In both aneurysms and dissections, this transformation to a proteolytic state likely plays an important pathophysiologic role in the development and progression of the aortic disease. The recognition of this pathophysiologic mechanism raises the potential for drug therapy to interrupt the cascade of events.

Context: Common fibrous histiocytoma (cFH) or dermatofibroma and dermatofibrosarcoma protuberans (DFSP) are 2 spindle cell mesenchymal tumors that are distinguished in part by their microscopic growth patterns and clinically by the greater propensity for DFSP to recur. Matrix metalloproteinases (MMPs) potentially play a role in modulating the growth patterns of cFH and DFSP by remodeling the extracellular matrix., Objective: To evaluate the immunohistochemical (IHC) expression of MMP-1, MMP-2, MMP-9, and MMP-14 in DFSP and cFH, because (1) MMP-1, MMP-2, MMP-9, and MMP-14 are synthesized by dermal fibroblasts, the major constituent of DFSP and cFH; and (2) platelet-derived growth factor B, which is overexpressed in most examples of DFSP because of t(17;22), activates ets-1, a transcription factor that regulates molecules associated with tumor invasion and metastasis, including MMP-1, MMP-3, and MMP-9., Design: Immunohistochemical studies were performed on archived, formalin-fixed, paraffin-embedded tissue of DFSP (n = 48) and cFH (n = 47).Results.-Significant IHC expression (>10% of tumor cells) in cFH included MMP-14 (27 [59%] of 46 tumors positive), MMP-2 (21 [47%] of 45 tumors positive), MMP-9 (9 [20%] of 45 tumors positive), and MMP-1 (6 [13%] of 46 tumors positive). No DFSPs showed significant IHC expression of any of the MMPs evaluated. However, anti- MMP-2 highlighted a rich microvascular element within deep tumor tissue present in 81% of DFSPs with a prominent subcutaneous component., Conclusion: Our IHC results indicate that MMP-1 and MMP-9 are not up-regulated in DFSP. Convincing expression of MMP-14 in cFH suggests that this MMP may affect the growth pattern of the lesion, perhaps by activating MMP-2 expression in tumor cells. In DFSP, MMP-2 may play a role in tumor angiogenesis.

We reported previously that low-density lipoprotein (LDL)-containing immune complexes (LDL-IC) stimulated matrix metalloproteinase-1 (MMP-1) expression in U937 histiocytes through Fc gamma receptor (FcgammaR)-mediated extracellular signal-regulated kinase pathway. The present study has explored the transcriptional mechanisms involved in the stimulation. Deletion analysis showed that LDL-IC stimulated MMP-1 promoter activity in cells transfected with the Construct 1 that contained a 4,334-bp MMP-1 promoter fragment, but had no effect in cells transfected with other constructs that had shorter MMP-1 promoter (2685-bp or less), suggesting that cis-acting elements located between -4334 and -2685 are required for the promoter stimulation. The mutation study further indicated that the activator protein-1 (AP-1) (-3471) or Ets (-3836) motifs in this distal region were essential for the LDL-IC-stimulated MMP-1 expression. Moreover, although above deletion analysis showed that LDL-IC did not stimulate MMP-1 promoter activity in cells transfected with constructs that contained the proximal AP-1 (-72) and Ets (-88) in the promoter fragments that are 2685-bp or less, the mutations of the -72 AP-1 or the -88 Ets motif in the construct 1 abolished the stimulation of MMP-1 expression by LDL-IC, suggesting that a long promoter sequence is required for the -72 AP-1 and -88 Ets motifs to be involved in the stimulation. Finally, electrophoretic mobility shift assay showed that LDL-IC stimulated the activities of transcription factors AP-1 and Ets. In conclusion, the present study shows that both the distal and proximal AP-1 and Ets motifs are required for LDL-IC-stimulated MMP-1 expression in U937 histiocytes.

Objective: To identify novel arthritis-associated and/or cartilage-specific self-epitopes recognized by T cells in patients with rheumatoid arthritis (RA)., Methods: Human analogs of several self-epitopes recognized in the rat adjuvant arthritis (AA) model (n = 13) were tested for T cell recognition in patients with RA and healthy controls. Recognition was assessed by proliferative activity of peripheral blood mononuclear cells (PBMC). In addition, cytokine production was determined., Results: Six out of the 13 peptides recognized during AA were also recognized by more than 20% of the RA patients, in contrast to only one out of the 16 control peptides that were not recognized during AA. The highest proliferative responses were to matrix metalloproteinase (MMP)-derived peptides. The response to a MMP-1 epitope was significantly higher in RA patients than in healthy controls. Moreover, this MMP-1 epitope increased interleukin 4 (IL-4) production of RA PBMC and decreased IL-4 production by control PBMC. The proliferative response to a MMP-3 epitope was similar in RA patients and controls; however, the MMP-3 epitope increased IL-4, and concomitantly IL-1beta and tumor necrosis factor-a production of RA PBMC, whereas these cytokines were unaffected in control PBMC., Conclusion: This study shows the presence of immune reactions to MMP-derived T cell epitopes that are associated with RA, suggesting a novel role of MMP in RA.

Systemic sclerosis is characterized by fibrosis and systemic autoimmunity; however, roles of autoantibodies in the development of fibrosis remain unknown in systemic sclerosis. The net accumulation of extracellular matrix is dependent on the balance between the synthesis and degradation of extracellular matrix components, the latter process regulated by matrix metalloproteinases. Matrix metalloproteinase-1 (interstitial collagenase-1) can initiate degradation of collagen types I-III that are major extracellular matrix constituents in affected skin of systemic sclerosis. In this study, we tested the hypothesis that systemic autoimmunity in systemic sclerosis induced anti-matrix metalloproteinase-1 autoantibodies that inhibited matrix metallo-proteinase-1 activity, resulting in collagen accumulation. Enzyme-linked immunosorbent assay using human recombinant matrix metalloproteinase-1 revealed that IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly elevated in sera from patients with systemic sclerosis, but not patients with active systemic lupus erythematosus or dermatomyositis, relative to normal controls. IgG anti-matrix metalloproteinase-1 autoantibody levels were significantly higher in patients with diffuse cutaneous systemic sclerosis than those found in patients with limited cutaneous systemic sclerosis. Furthermore, IgG anti-matrix metalloproteinase-1 antibody levels significantly correlated with the extent of fibrosis in the skin, lung, and renal blood vessels. The presence of IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients was confirmed by immunoblotting analysis. Remarkably, IgG anti-matrix metalloproteinase-1 autoantibody in sera from systemic sclerosis patients inhibited matrix metalloproteinase-1 collagenase activity. Collectively, the results of this study suggest that anti-matrix metalloproteinase-1 autoantibody contributes to the development of fibrosis by inhibiting matrix metalloproteinase-1 collagenase activity and reducing the extracellular matrix turnover and suggest that the presence of anti-matrix metalloproteinase-1 autoantibody in systemic sclerosis is the link between systemic autoimmunity and fibrosis.

Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Here, we show that collagenase activity promotes bone resorption by activating adherent osteoclasts to resorb bone. Inhibition of interstitial collagenase activity, either with peptidomimetic hydroxymates or with a specific anti-interstitial collagenase inhibiting antibody, reduced bone resorption by 73-92%. Equal numbers of osteoclasts adhered to bone in the presence of collagenase inhibitors and osteoclast survival was unaffected. In contrast, formation of actin rings and polarization of the vacuolar-H+-ATPase (V-ATPase) to ruffled membranes, two indicators of osteoclast activation, were decreased by inhibiting collagenase activity and stimulated in the presence of cleaved or heat-denatured type I collagen in proportion to increases and decreases of bone resorptive activity. Addition of excess recombinant osteoprotegerin-ligand to cultures did not restore bone resorption in the presence of interstitial collagenase inhibitors. These data support the hypothesis that cleaved collagen stimulates osteoclastic bone resorption by triggering cytoskeletal reorganization and transport of V-ATPase from cytoplasmic stores to ruffled membranes.

Objective: To determine the sites of cleavage and denaturation of type II collagen (CII) by collagenase(s) in healthy and osteoarthritic (OA) human articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 (MMP-1) and MMP-13., Methods: Single (per subject) full-depth specimens from femoral condylar cartilage were isolated from articulating surfaces at autopsy from 8 subjects without arthritis and during arthroplasty from 10 patients with OA. Fixed frozen sections of cartilage were examined by immunoperoxidase localization, using antibodies to the collagenase-generated cleavage site in CII, to an intrachain epitope recognized only in denatured CII, and to MMP-1 and MMP-13 (proenzyme, activated enzyme, or enzyme/inhibitor complex)., Results: Staining for collagen cleavage, denaturation, and both MMPs was weak to moderate and was frequently observed in pericellular sites in cartilage from younger, nonarthritic subjects. In specimens from older subjects, this staining was often more widespread and of greater intensity. Similar staining was usually, but not always, seen for all antibodies. In OA cartilage, staining was often stronger and more intense than that in normal cartilage from older subjects, and the distribution of staining was often similar for the different antibodies. Pericellular staining in the deep zone was frequently more pronounced in arthritic cartilage and extended to territorial and sometimes interterritorial sites. In very degenerate specimens, staining was distributed throughout most of the cartilage matrix., Conclusion: These observations provide evidence for the presence of limited cleavage and denaturation of CII restricted to mainly pericellular and superficial sites in cartilage from younger, healthy subjects, where MMP-1 and MMP-13 are also selectively localized. Collagen degradation is more extensive and often more pronounced in cartilage from older, nonarthritic subjects. Characteristic changes in early OA are similar to those seen with aging in cartilage from older, healthy subjects, with collagen damage and collagenases concentrated closer to the articular surface. There was usually a close correspondence between the cleavage and denaturation of CII and the sites at which these collagenases were detected, suggesting that both MMPs are involved in the physiology and pathology. There was no evidence that the damage to CII is ordinarily initiated in sites other than at and near the articular surface and around chondrocytes.

We reported recently that immune complexes (ICs) induced matrix metalloproteinase-1 (MMP-1) expression in U937 histiocytes. The present study was undertaken to determine the effect of pretreatment of U937 cells with interferon-gamma (IFN-gamma) on IC-induced MMP-1 expression. Our flow cytometry studies showed that IFN-gamma upregulated the surface expression of FcgammaRI, but not FcgammaRII. Results also showed that pretreatment of the cells with IFN-gamma augmented LDL-containing IC (LDL-IC)-induced MMP-1 secretion in a dose- and time-dependent manner. Furthermore, Northern blot analysis revealed that IFN-gamma pretreatment led to a marked increase in MMP-1 mRNA. Finally, we demonstrated that PD98059 was able to block LDL-IC-induced MMP-1 secretion, regardless of whether the cells were pretreated with IFN-gamma or not, suggesting that IFN-gamma pretreatment did not alter the essential role of the ERK signaling pathway in LDL-IC-induced MMP-1 expression. In conclusion, the present study has demonstrated that IFN-gamma pretreatment augments LDL-IC-induced MMP-1 expression in U937 cells, thus elucidating an immune mechanism potentially involved in plaque destabilization., (Copyright 2001 Elsevier Science (USA).)

Here, we describe a new function for plasmin and matrix metalloproteinases (MMPs), which is to regulate the regression of capillary tubes in three-dimensional extracellular matrix environments. Using a well-described capillary morphogenesis system in three-dimensional collagen matrices, a new model of capillary regression has been established by adding plasminogen to the culture medium. Plasminogen is converted to plasmin by endothelial cell plasminogen activators which then induces matrix metalloproteinase-dependent collagen gel contraction and capillary regression. Plasminogen addition results in activation of MMP-1 and MMP-9, which then results in collagen proteolysis followed by capillary regression. The endothelial cells undergo apoptosis following gel contraction as detected by flow cytometric analysis as well as by detectable caspase-3 cleavage and caspase-dependent cleavage of the actin cytoskeletal regulatory protein, gelsolin. In addition, directly correlating with the contraction response, tyrosine phosphorylation of p130cas, an adapter protein in the focal adhesion complex, is observed followed by disappearance of the protein. Proteinase inhibitors that block MMPs (TIMP-1 or TIMP-2), plasminogen activators (PAI-1) or plasmin (aprotinin) completely block the gel contraction and regression process. In addition, chemical inhibitors of MMPs that block capillary regression also block MMP-1 and MMP-9 activation suggesting that a key element in this regression response is the molecular control of MMP activation by endothelial cells. Blocking antibodies directed to MMP-1 or MMP-9 interfere with capillary regression while blocking antibodies directed to PAI-1 accelerate capillary regression suggesting that endogenous synthesis of PAI-1 negatively regulates this process. These data present a novel system to study a new mechanism that may regulate regression of capillary tubes, namely, plasmin and MMP-mediated degradation of extracellular matrix.