Search

The human precorneal tear film is a special body fluid, since it is a complex mixture of proteins, lipids, small bioactive molecules, and their concentrations and relative distribution represent not only the metabolic state of the ocular surface but also the systemic and local homeostasis of the outer eye and the human body. This suggests that biochemical analysis of the precorneal tear film composition may provide a non-invasive tool for diagnosis and monitoring of disease progression or treatment efficacy in human medicine. However, collecting tears is demanding, and obtaining reproducible and unaltered samples is challenging because of the small sample volumes of tears. Several methods are available for tear collection as a preparatory step of precorneal tear film analysis, and the collection method used has to be assessed since it has a critical impact on the effectiveness of the assays and on the quality of the results. Each sampling method has advantages and disadvantages; therefore, it is not easy to choose the appropriate collecting method for tear collection. To overcome these limitations various methods have been recommended by different authors for special aspects of specific tests. The aim of our review was to evaluate tear sampling methods with regard to our ongoing biochemical analysis. *Contributed equally.

Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

Background Systemic sclerosis (SSc) is an autoimmune disease, characterized by widespread small vessel vasculopathy, immune dysregulation with production of autoantibodies, and progressive fibrosis. SSc may be associated with sicca syndrome. Changes in levels of proangiogenic and proinflammatory cytokines had already been determined largely in serum, however, the local inflammatory and cytokine milieu in the tear of SSc patients has not yet been evaluated. Objectives We wished to determine VEGF and other cytokine and chemokine levels in tear samples of SSc patients. Methods First, forty-three patients (40 female and 3 men, mean (SD) age 61 (48–74) years) with SSc and 27 healthy controls were enrolled in the VEGF study. Basal tear sample collection and tear velocity investigations were carried out followed by an ophthalmological examination. Total protein concentrations and VEGF levels were determined in tear samples. In the multiple cytokine study, unstimulated tear samples were collected from nine patients with SSc and 12 age- and gender-matched healthy controls. The relative levels of 102 different cytokines were determined by a cytokine array, and then absolute levels of four key cytokines were determined by a magnetic bead assay. Results In the first study, the mean collected tear fluid volume developed 10.4 L (1.6–31.2) in patients and 15.63 L (3.68–34.5) in control subjects. The mean total protein level was 6.9 g/L (1.8–12.3) and 4.1 g/L (0.1–14.1) in tear samples of SSc patients and controls, respectively. In patients with SSc, the mean VEGF tear concentration was 4.9 pg/L (3.5–8.1) compared to 6.15 pg/L (3.84–12.3) in healthy samples. Multicytokine-array studies revealed shifted cytokine profile characterized by predominance of proinflammatory mediators in the tear samples of SSc patients. Out of the 102 analyzed proteins, nine were significantly increased in tears of patients with SSc. Based on the multiplex bead results, CRP, interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (mcp-1) levels were significantly higher in tears of patients with SSc compared to controls. Conclusions Impaired angiogenesis has been found by other investigators in SSc. This is reflected by lower VEGF levels in the tear samples of SSc patients compared to controls. The multi-cytokine array study revealed increased production of CRP and two important pro-inflammatory chemokines in the tear of SSc patients. Our current data depict a group of inflammatory mediators, which may play a significant role in ocular pathology of SSc. Disclosure of Interest None declared

The thymus provides the microenvironment in which thymocytes develop into mature T cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize the peptide-loaded self-MHC molecules found in the thymus, and therefore lack a TCR signal, undergo a default death pathway named "death by neglect" in the thymic cortex. In the absence of this TCR signaling, it has been suggested that binding of glucocorticoids to - or the ligation of certain cell surface molecules, such as CD8, CD24, CD45, or CD99 on - these neglected thymocytes will induce them to enter the apoptotic program. Apoptotic thymocytes are cleared by the surrounding macrophages and, as a consequence, these macrophages are known to release various molecules, such as adenosine, retinoids, TGF-β, ATP, and carbon monoxide. Interestingly, all these molecules have been described to induce or promote apoptosis in thymocytes in the absence of TCR signaling. Here, we propose that thymic macrophages, because they continually engulf apoptotic cells, might constantly provide these cell death-inducing signals, and thus contribute to the formation of a thymic milieu that ensures the effective induction of "death by neglect".

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. It acts as a G protein in transmembrane signaling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion, and cell death. Though many biological functions of the enzyme have already been described or proposed previously, studies of TG2 null mice by our laboratory during the past years revealed several novel in vivo roles of the protein. In this review we will discuss these novel roles in their biological context.

Although serious ocular manifestations of systemic sclerosis (SSc) have been described, tear analysis of patients with SSc has not been performed in previous studies. Our aim was to measure a wide panel of cytokines and chemokines in tears of patients with SSc and to assess the most significant molecules with a more sensitive and specific method. Unstimulated tear samples were collected from nine patients with SSc and 12 age- and gender-matched healthy controls. The relative levels of 102 different cytokines were determined by a cytokine array, and then absolute levels of four key cytokines were determined by a magnetic bead assay. Array results revealed shifted cytokine profile characterized by predominance of inflammatory mediators. Of the 102 analyzed molecules, nine were significantly increased in tears of patients with SSc. Based on the multiplex bead results, C-reactive protein, interferon-γ-inducible protein-10, and monocyte chemoattractant protein-1 levels were significantly higher in tears of patients with SSc. Our current data depict a group of inflammatory mediators, which play a significant role in ocular pathology of SSc; furthermore, they might function as excellent candidates for future therapeutic targets in SSc patients with ocular manifestations.

Background. Systemic sclerosis is an autoimmune disease, characterized by widespread small vessel vasculopathy, immune dysregulation with production of autoantibodies, and progressive fibrosis. Changes in levels of proangiogenic cytokines had already been determined largely in serum. Our aim was to assess the levels of VEGF in human tears of patients with SSC.Patients and methods. Forty-three patients (40 female and 3 men, mean (SD) age 61 (48–74) years) with SSc and 27 healthy controls were enrolled in this study. Basal tear sample collection and tear velocity investigations were carried out followed by an ophthalmological examination. Total protein concentrations and VEGF levels were determined in tear samples.Results. The average collected tear fluid volume developed 10.4 μL (1.6–31.2) in patients and 15.63 μL (3.68–34.5) in control subjects. The average total protein level was 6.9 μg/μL (1.8–12.3) in tears of patients and control tears contained an average of 4.132 μg/μL (0.1–14.1) protein. In patients with SSc the average concentration of VEGF was 4.9 pg/μL (3.5–8.1) and 6.15 pg/μL (3.84–12.3) in healthy samples.Conclusions. Total protein production was increased because of the smaller tear volume. Decreased VEGF in tear of SSc patients can be explained also by the decreased tear secretion of patients.

Introduction Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. Methods Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGF-β1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model. Results TG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-β1, IL-1β and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-β1, but increased that of IL-1β and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1β and TNF-α, but significantly enhanced the TGF-β1 production. Neutralizing TGF-β antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-β, but in an increased IL-1β and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1β production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-β suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model. Conclusions These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-β1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0592-7) contains supplementary material, which is available to authorized users.

Timed initiation of apoptotic cell death followed by efficient removal mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Besides phagocytosis, clearance of apoptotic cells also involves suppression of inflammatory responses by apoptotic cells mediated by both direct inhibition of pro-inflammatory cytokine production and release of soluble anti-inflammatory factors, which act in a paracrine or autocrine fashion to amplify or sustain the anti-inflammatory response. Previous work has demonstrated that during engulfment of apoptotic cells adenosine is produced in sufficient amounts to trigger both adenosine A2A receptors (A2ARs) and A3 receptors (A3Rs). Adenosine bound to A2ARs of macrophages activated the adenylate cyclase pathway to suppress the apoptotic-cell induced, NO-dependent formation of neutrophil migration factors. Here we show by using A3R null engulfing macrophages that the adenosine produced triggers the A3Rs as well, which attenuate the A2AR signaling by inhibiting adenylate cyclase. As a result, the balance in the activation of A2ARs and A3Rs determines the amounts of NO and consequently the levels of neutrophil chemoattractants formed. Since during phagocytosis of apoptotic cells the expression of A2ARs increases, while that of A3Rs decreases, on long term adenosine suppresses the proinflammatory responses in engulfing macrophages.

Fatty acid concentrations, in particular n-3 and n-6 polyunsaturated fatty acids (PUFAs), have been described to be dysregulated in atopic dermatitis (AD) patients. The role of genetic polymorphisms of fatty acid enzymes in AD is controversial. We determined in a Hungarian cohort of healthy volunteers (n = 20) and AD patients (n = 20) triglyceride-, sterol- and phospholipid-bound fatty acids in the plasma, mRNA expression of fatty acid desaturase 2 (FADS2) and stearoyl-coenzyme A desaturase 1 in peripheral blood mononuclear cells (PBMCs) and FADS2 concentrations in plasma. We observed higher levels of monounsaturated fatty acids, 16:1 versus 16:0 ratios in phospholipids, triglycerides and sterol esters in patients compared to healthy subjects. In addition higher levels of the FADS2-derived n-6 PUFAs γ-linolenic acid and dihomo-γ-linolenic acid were observed in PBMCs of patients as well as lower levels of n-3 PUFAs. We conclude that the increased expression of FADS2 in PBMCs, as a representative tissue accessible from human blood of AD patients, might be responsible for higher levels of FADS2-derived n-6 PUFAs and lower n-3 PUFA levels in patients.

The thymus provides the microenvironment in which thymocytes develop into mature T cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize the peptide-loaded self-MHC molecules found in the thymus, and therefore lack a TCR signal, undergo a default death pathway named "death by neglect" in the thymic cortex. In the absence of this TCR signaling, it has been suggested that binding of glucocorticoids to - or the ligation of certain cell surface molecules, such as CD8, CD24, CD45, or CD99 on - these neglected thymocytes will induce them to enter the apoptotic program. Apoptotic thymocytes are cleared by the surrounding macrophages and, as a consequence, these macrophages are known to release various molecules, such as adenosine, retinoids, TGF-β, ATP, and carbon monoxide. Interestingly, all these molecules have been described to induce or promote apoptosis in thymocytes in the absence of TCR signaling. Here, we propose that thymic macrophages, because they continually engulf apoptotic cells, might constantly provide these cell death-inducing signals, and thus contribute to the formation of a thymic milieu that ensures the effective induction of "death by neglect".

Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble >find me> signals, 2) recognition and phagocytosis via cell surface-presenting >eat me> signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor d. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation. Copyright © 2011 by The American Association of Immunologists, Inc., This work was supported by Hungarian grants from the National Research Fund (K77587, TS-44798, and F67632).

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.