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Regulatory T cells (Tregcells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Tregcells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Tregcells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Tregcells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Tregcell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62LloTregcell subset that is distinct from previously characterized intestinal Tregcell populations as the main IL-27 producers. Collectively, our study uncovers a new Tregcell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Tregcell-mediated immune regulation.

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL(CD62L) and CCR7. Interestingly, we discovered that while SalmonellaTyphimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+T-bet+ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+T-bet+ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines.

Innate lymphoid cells (ILCs) are enriched in mucosae and have been described as tissue-resident. Interestingly, ILCs are also present within lymph nodes (LNs), in the interfollicular regions, the destination for lymph-migratory cells. We have previously shown that LN ILCs are supplemented by peripheral tissue-derived ILCs. Using thoracic duct cannulations, we here enumerate the intestinal lymph ILCs that traffic from the intestine to the mesenteric LNs (MLNs). We provide, for the first time, a detailed characterisation of these lymph-migratory ILCs. We show that all ILC subsets migrate in lymph, and while global transcriptional analysis reveals a shared signature with tissue-resident ILCs, lymph ILCs express migration-associated genes including S1PRs, SELL(CD62L) and CCR7. Interestingly, we discovered that while SalmonellaTyphimurium infections do not increase the numbers of migrating ILCs, infection changes their composition and cytokine profile. Infection increases the proportions of RORyt+T-bet+ILCs, levels of IFNγ, and IFNγ/GM-CSF co-expression. Infection-induced changes in migratory ILCs are reflected in colon-draining MLN ILCs, where RORyt+T-bet+ILCs accumulate and display corresponding increased cytokine expression. Thus, we reveal that ILCs respond rapidly to intestinal infection and can migrate to the MLN where they produce cytokines.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Loss of epithelial barrier integrity is implicated in a number of human lung diseases. However, the molecular pathways underlying this process are poorly understood. In a phenotypic screen, we identified Axl kinase as a negative regulator of epithelial phenotype and function. Furthermore, suppression of Axl activity by a small molecule kinase inhibitor or downregulation of Axl expression by small interfering RNA led to: (1) the increase in epithelial surfactant protein expression; (2) a cell morphology transition from front-rear polarity to cuboidal shape; (3) the cytoskeletal re-organization resulting in decreased cell mobility; and (4) the acquisition of epithelial junctions. Loss of Axl activity reduced activation of the Axl canonical pathway members, Akt and extracellular signal-regulated kinase-1/2 and resulted in the loss of gene expression of a unique profile of epithelial-to-mesenchymal transition transcription factors including SNAI2, HOXA5, TBX2or TBX3. Finally, we observed that Axl was activated in hyperplasia of epithelial cells in idiopathic pulmonary fibrosis where epithelial barrier integrity was lost. These results suggest that the Axl kinase signaling pathway is associated with the loss integrity of alveolar epithelium in pathological remodeling of human lung diseases.

Loss of epithelial barrier integrity is implicated in a number of human lung diseases. However, the molecular pathways underlying this process are poorly understood. In a phenotypic screen, we identified Axl kinase as a negative regulator of epithelial phenotype and function. Furthermore, suppression of Axl activity by a small molecule kinase inhibitor or downregulation of Axl expression by small interfering RNA led to: (1) the increase in epithelial surfactant protein expression; (2) a cell morphology transition from front-rear polarity to cuboidal shape; (3) the cytoskeletal re-organization resulting in decreased cell mobility; and (4) the acquisition of epithelial junctions. Loss of Axl activity reduced activation of the Axl canonical pathway members, Akt and extracellular signal-regulated kinase-1/2 and resulted in the loss of gene expression of a unique profile of epithelial-to-mesenchymal transition transcription factors including SNAI2, HOXA5, TBX2 or TBX3. Finally, we observed that Axl was activated in hyperplasia of epithelial cells in idiopathic pulmonary fibrosis where epithelial barrier integrity was lost. These results suggest that the Axl kinase signaling pathway is associated with the loss integrity of alveolar epithelium in pathological remodeling of human lung diseases.

Recent findings suggest that β‐adrenergic blockers have antinociceptive properties. The aim of this study was to compare levels of large‐joint pain between those taking adrenergic blockers and those taking other antihypertensive medications. Data from the Genetics of Osteoarthritis and Lifestyle (GOAL) study, a secondary‐care cohort of osteoarthritis (OA) patients, were used. Joint pain was assessed using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain scores in 873 individuals with symptomatic hip and/or knee OA and hypertension, who were taking ≥1 prescription antihypertensive medications. The association between adrenergic blocker prescription and at least moderate joint pain (WOMAC score <75) and use of prescription analgesics was examined using binary logistic regression. Analyses were adjusted for age, sex, body mass index, knee or hip OA, history of joint replacement (at other joints), anxiety, and depression. The use of β‐adrenergic blockers was associated with lower WOMAC pain scores and with a lower prevalence of joint pain after adjustment for demographic variables and comorbidity (adjusted odds ratio [ORadj] for pain 0.68 [95% confidence interval (95% CI) 0.51, 0.92]; P< 0.011). No associations with pain were observed with use of alpha‐blockers (ORadjfor pain 0.94 [95% CI 0.55, 1.58]) or with any other class of antihypertensive medications. Prescription of beta‐blockers was also associated negatively with opioid use (ORadjfor opioids 0.73 [95% CI 0.54, 0.98]; P< 0.037) and with the use of prescription analgesics in general (ORadjfor analgesics 0.74 [95% CI 0.56, 0.94]; P< 0.032). The use of beta‐blockers is associated with less joint pain and a lower use of opioids and other analgesics in individuals with symptomatic large‐joint OA. This observation needs to be confirmed by other studies.

Objective.To analyze the 3-D bone area from an osteoarthritis (OA) cohort demonstrating no change in cartilage thickness.Methods.Twenty-seven women with painful medial knee OA had magnetic resonance images at 0, 3, and 6 months. Images were analyzed using active appearance models.Results.At 3 and 6 months, the mean change in medial femoral bone area was 0.34% (95% CI 0.04–0.64) and 0.61% (95% CI 0.32–0.90), respectively. Forty-one percent of the subjects had progression greater than the smallest detectable difference at 6 months.Conclusion.In this small cohort at high risk of OA progression, bone area changed at 3 and 6 months when cartilage morphometric measures did not.

Osteoarthritis (OA) is the most common joint disorder in the world and represents the leading cause of pain and disability in the elderly population. Advancing age remains the single greatest risk factor for OA. Several studies have characterised disease development in the guinea pig ageing model of OA in terms of its joint histopathology and inflammatory cytokine profile. However, the quadriceps muscle has yet to be studied in relation to age-related disease onset or early disease progression. Therefore, we examined whether the initiation of OA in the Dunkin Hartley guinea pig is associated with changes in the quadriceps skeletal muscle. Male Dunkin Hartley guinea pigs (N = 24) were group housed with free access to standard guinea pig chow and water. At 2, 3, 5 and 7 months of age, six animals were selected based on their proximity to the median weight of the cohort. OA severity was graded at each time point by the assessment of toluidine blue stained step coronal sections of the total knee joint. Serum CTX II was measured as a potential biomarker of OA severity. Myosin Heavy Chain (MHC) isoforms were determined by a validated real-time PCR assay. Oxidative and glycolytic potential was determined in quadriceps homogenates via the measurement of ICDH and LDH activity. Initiation of OA in the DH strain guinea pig occurred between 2 and 3 months of age and progressed until 7 months when the final analyses were conducted. Serum CTX II significantly decreased during this early period of OA initiation and levels were unrelated to the histopathological severity of knee OA at any of the time points assessed. MHC mRNA measurements revealed a significant elevation in MHC IIX mRNA (associated with fast-twitch skeletal muscle fibres) coincident with the initiation of OA at 3 months of age, with preliminary findings suggestive of a positive correlation to OA severity at this time point. These preliminary findings suggest that disease initiation in the ageing guinea pig model of OA is not associated with overt quadriceps muscle atrophy but instead is coincident with altered expression of mRNAs associated with quadriceps skeletal muscle contractile properties (specifically fast-twitch MHC IIX).

ObjectiveTo determine whether selfreported early adult life malalignment of knees or feet are risk factors for knee or hip osteoarthritis OA.MethodsParticipants in the Genetics of Osteoarthritis and Lifestyle case–control database were sent a questionnaire n 3,022 containing linedrawing instruments for selfreported knee and foot alignment at ages 20–29 years. Respondents were categorized as having straight, valgus, or varus knee, and straight, toein, or toeout feet. Radiographic criteria were used to define current isolated knee or hip OA, combined knee and hip OA, or nonOA controls. Odds ratios ORs, adjusted ORs, and 95 confidence intervals 95 CIs were calculated and logistic regression was performed.ResultsThe response rate was 72; 87.5 of responders n 1,901 completed the alignment questions. Increased risk of isolated knee OA occurred with early adult varus adjusted OR 5.16, 95 CI 2.87–9.41 and valgus knees adjusted OR 3.16, 95 CI 1.04–9.64. The positive association between knee OA and toein foot was explained by varus knee. There was an increased risk of combined kneehip OA from varus adjusted OR 4.52, 95 CI 2.39–8.53 and valgus knees adjusted OR 3.07, 95 CI 0.99–9.54. Varus knee was associated with risk of medial tibiofemoral OA, whereas valgus knee was associated with risk of lateral tibiofemoral and lateral patellofemoral OA. Toeout foot was associated with reduced medial patellofemoral OA. For knee OA, a multiplicative interaction analysis between occupational risks and varusvalgus yielded an ORintof 3.20 95 CI 1.08–9.49.ConclusionConstitutional alignment of the leg in terms of varus or valgus knee or foot rotation may be a significant factor in determining development and distribution of knee but not hip OA.

ADAMs are membrane‐anchored glycoproteins containing a disintegrin and metalloprotease domain that have important roles in fertilization, development, and diseases such as Alzheimer's dementia. Here we present the first evidence for catalytic activity of ADAM28, a protein that is highly expressed in the epididymis and lymphocytes. Recombinant ADAM28 cleaves myelin basic protein at two sites. The catalytic activity of ADAM28 is not sensitive to tissue inhibitors of metalloproteases 1 and 2, but can be abolished by a mutation in the catalytic site. Catalytically active ADAM28 will be valuable for further studies of its role in sperm maturation and lymphocyte function.

ADAMs are membrane-anchored glycoproteins containing a disintegrin and metalloprotease domain that have important roles in fertilization, development, and diseases such as Alzheimer’s dementia. Here we present the first evidence for catalytic activity of ADAM28, a protein that is highly expressed in the epididymis and lymphocytes. Recombinant ADAM28 cleaves myelin basic protein at two sites. The catalytic activity of ADAM28 is not sensitive to tissue inhibitors of metalloproteases 1 and 2, but can be abolished by a mutation in the catalytic site. Catalytically active ADAM28 will be valuable for further studies of its role in sperm maturation and lymphocyte function.

We have expressed G1-G2 mutants with amino acid changes at the DIPEN341↓342FFGVG and ITEGE373↓374ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN341to DIGSA341partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation 342FFGVG to342GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF343, were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation 374ARGSV to374NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn341↓Phe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA341mutant but absent in the wild-type,342GTRVG, 374NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase andvice versa. Aggrecanase-derived G1 domains with ITEGE373C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu373↓Ala bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN341G1 domain may be important for regulating aggrecanase cleavage.

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 (‘a disintegrin and metalloprotease 28’), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.

Our objective was to test the hypothesis that diurnal changes occur in thickness or volume of the femoral articular cartilage of the knee in asymptomatic young adults. Fat‐suppressed three‐dimensional (3D) spoiled gradient‐echo magnetic resonance imaging (MRI) was employed. Six volunteers each were scanned early in the morning and at the end of a working day spent mainly standing. This protocol was repeated on 3 successive weeks. Femoral cartilage volumes were obtained via semiautomatic segmentation that employed a seeding algorithm. These segmentations then were regridded onto a 500‐pixel template, and differences in the resulting thickness maps were assessed. Analysis of variance showed no significant diurnal variation in overall volume or thickness. The reproducibility for volume (test‐retest coefficient of variation) was 1.6%. There were, however, statistically‐significant diurnal changes in the thickness maps. Cartilage thickness decreased by up to 0.6 mm during the day in each of the following three specific locations: the patellofemoral compartment, the lateral tibiofemoral compartment, and the medial tibiofemoral compartment. Elsewhere, cartilage thickness was unchanged or increased by up to 0.5 mm. We conclude that, in asymptomatic young adults, cartilage volume does not change during the day; however, the cartilage does become thinner in locations that encounter the greatest biomechanical force. Magn Reson Med 43:126–132, 2000. © 2000 Wiley‐Liss, Inc.

Metalloprotease disintegrins (adisintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin,cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- andphoxhomology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37°C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.

We have investigated the susceptibility of both the helical and non‐helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37°C and pH values from 3.5 to 7.0. Cleavage occurs only within the non‐helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Chondrocalcinosis (CC) most commonly results from calcium pyrophosphate crystal deposition (CPPD). The objective of this study is to examine the association between candidate single-nucleotide polymorphisms (SNPs) and radiographic CC.

The objective was to investigate potential gene-environment interaction between body mass index (BMI) and each of eight TGFβ1 polymorphisms in knee and hip osteoarthritis (OA).

We aimed to describe the distribution of radiographic chondrocalcinosis (CC) and to examine whether metacarpophalangeal joint (MCPJ) calcification and CC at other joints occurs in the absence of knee involvement.

Although the presence of bone marrow lesions (BMLs) on magnetic resonance images is strongly associated with osteoarthritis progression and pain, the underlying pathology is not well established. The aim of the present study was to evaluate the architecture of subchondral bone in regions with and without BMLs from the same individual using bone histomorphometry.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at θ = 90° and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength μ = 0.45) or 0.53 M NaCl (μ = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (μ = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly (Arg) from DNA higher ionic strength using NaCl, MgCl2 or CaCl2 similar ionic strength using NaClO4 and lower ionic strength using Na2SO4 was needed then to dissociate poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.