Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells.

OBJECTIVES: A gene-environment interaction between HLA-DR shared epitope genes and smoking in anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis (RA) has been reported. Identification of citrullinated proteins in bronchoalveolar lavage (BAL) cells from smokers has led to the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA. OBJECTIVE: To confirm and extend these findings. METHODS: Immunohistochemistry was performed on BAL cells and bronchial mucosal biopsy sections obtained through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. Two antibodies recognising citrullinated proteins, two antibodies recognising peptidylarginine deiminase (PAD)2 enzyme and one recognising PAD4 enzyme were used. RESULTS: Citrullinated proteins are upregulated in BAL cells of healthy smokers compared with healthy non-smokers. This was associated with higher expression of the PAD2 enzyme. The same level of citrullinated proteins was present in bronchial mucosal biopsy specimens of healthy smokers and non-smokers, despite higher expression of PAD2 in smokers. CONCLUSION: This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

Cellular distribution of the C-type II lectin dendritic cell immunoreceptor (DCIR) and its expression in the rheumatic joint: identification of a subpopulation of DCIR+ T cells.

OBJECTIVE: An association to variations in the dendritic cell immunoreceptor (DCIR) gene with rheumatoid arthritis (RA) was recently shown. However, protein expression of DCIR has so far not been assessed in a disease setting. In the present work, we aimed to determine the cellular and tissue distribution of this receptor in healthy controls and in patients with RA before and after local glucocorticoid administration. METHODS: DCIR mRNA expression was evaluated by quantitative PCR (n=3) and protein expression by flow cytometry (n=18), immunohistochemistry (n=14) and double immunofluorescence (n=5). RESULTS: DCIR protein was not detected in healthy synovia. By contrast, expression was abundant on cells from rheumatic joints in synovial fluid and in tissue. Following corticosteroid treatment this expression was downregulated. Interestingly, DCIR could be detected on natural killer (NK) cells and T cells, and CD4+ and CD8+, as well as on monocytes, B cells, dendritic cells and granulocytes. The frequency of DCIR+ T cells and the level of surface expression were increased in the rheumatic joint compared to blood. In synovial fluid the typical DCIR+ T cells were large activated cells, whereas blasted DCIR+ T cells were not detected in blood. CONCLUSIONS: We demonstrate increased protein and mRNA expression of DCIR in RA, especially in the rheumatic joint. Expression was widespread and included a subpopulation of T cells. This suggests that the inflammatory synovial environment induces DCIR expression, and this may be related to synovial T cell function. Ligation of DCIR, or lack thereof, could contribute to the chronic inflammation characterising autoimmune diseases such as RA.

Down-regulation of interferon-gamma parallels clinical response to selective leukocyte apheresis in patients with inflammatory bowel disease: a 12-month follow-up study.

BACKGROUND & AIMS: Pilot studies have indicated a therapeutic role for an apheresis device (Adacolumn) that selectively adsorbs leukocytes in patients with inflammatory bowel diseases. It may also exert immunoregulatory effects contributing to its clinical efficacy. This study aimed to correlate the clinical response to leukocyte apheresis with the expression of key cytokines in mucosal tissue, in peripheral leukocytes, and in plasma. METHODS: Ten patients (seven with Crohn's disease and three with ulcerative colitis, median age: 31 years) with mild to moderately chronic activity were recruited to an open study. Patients were refractory to or had a relapse despite conventional treatment including azathioprine. Leukocyte apheresis was performed once a week for five consecutive weeks. Clinical efficacy was assessed on week 7 and after 12 months. Colonoscopy with multiple biopsies was performed at the start of the study and after 7 weeks for semiquantitative immunohistochemical analyses of cytokines. Cytokine levels in blood and the proportion of cytokine producing CD4+ and CD8+ lymphocytes were determined. RESULTS: The apheresis procedures were well tolerated and no major adverse events were encountered. The median clinical activity score decreased from 12 to 7 on week 7 (P=0.031, n=9) and to 4 after 12 months (P=0.004, n=9). Five patients were in clinical remission at the 12th month. Tissue interferon (IFN)-gamma-positive T-cells decreased in clinical responders (P=0.027) after apheresis. In parallel, significantly lower levels of IFN-gamma-producing lymphocytes were detected in peripheral blood. IFN-gamma-positive cells in pretreatment biopsies completely disappeared or decreased in posttreatment biopsies sampled on week 7 in responders (P=0.027) and appeared to predict the maintenance of long-term remission or response after 12 months. CONCLUSIONS: Leukocyte apheresis is a novel and safe nonpharmacological adjunct therapy that may prove useful in steroid refractory or dependent patients when conventional drugs have failed. Down-regulation of IFN-gamma in mucosal biopsies and in peripheral leukocytes may be a predictive marker for sustained, long-term response.

Citrullination is an inflammation-dependent process.

OBJECTIVES: To study the presence of citrullinated proteins in inflammatory conditions and in clinically non-affected tissues of controls. METHODS: Synovial biopsy specimens from 19 patients with rheumatoid arthritis and 10 healthy controls were investigated by immunohistochemistry. Additionally, muscle tissue from 5 patients with polymyositis and from 7 healthy controls, intestinal tissue from macroscopically affected and non-affected areas from 10 patients with inflammatory bowel disease (IBD) and tonsil tissues from 4 chronically inflamed tonsils were studied. RESULTS: Citrullinated proteins were present in all synovial biopsy specimens from patients with rheumatoid arthritis, whereas only three of 10 healthy synovial biopsy specimens showed scarce amounts of citrullination. Citrullination was also present in all myositis-affected muscles, whereas it was absent in the muscle tissues of controls. All tonsil biopsy specimens studied were positive for citrulline. Even though more frequently detected in the macroscopically affected colonic areas, no marked difference was observed in the pattern or extent of citrullination between the macroscopically affected and non-affected intestinal IBD tissues. CONCLUSION: Citrullination is present in a wide range of inflammatory tissues, suggesting that this process is inflammation dependent rather than disease dependent.

Standardisation of synovial tissue infiltrate analysis: how far have we come? How much further do we need to go?

Changes in cellular infiltrate and expression of cytokines, chemokines, and cell adhesion molecules as a result of therapeutic interventions in rheumatoid arthritis can be demonstrated in the synovial membrane. However, before synovial tissue analysis can be used as an outcome measure in such studies, standardisation of the site and method of synovial tissue acquisition, methods of tissue processing, and appropriate methods of detection and measurement of cell lineage specific markers and relevant biological proteins is needed.

Nitric oxide formation in the oropharyngeal tract: possible influence of cigarette smoking.

Cigarette smoking reduces the level of nitric oxide (NO) in exhaled air by an unknown mechanism. The view that part of the effect of cigarette smoking on NO production should occur in the oropharyngeal tract is supported by several studies. We have therefore compared smokers and non-smokers regarding non-enzymatic formation of NO from nitrite in the oral cavity since this is a primary candidate target for cigarette smoke. We have also looked at NO synthase-dependent NO formation in the mucosa of the oropharyngeal tract as an alternative target for the inhibitory effect induced by cigarette smoke. Smokers exhaled 67% lower levels of NO than controls (p<0.01, n=15 each group). We could not detect any significant difference in salivary nitrite, nitrate or ascorbate between smokers and non-smokers. Mouthwash with the antibacterial agent chlorhexidine reduced salivary nitrite (-65%) and exhaled NO levels (-10%) similarly in the two groups. Immunohistochemical techniques revealed dense expression of inducible (but not endothelial or neuronal) NO synthase in the squamous epithelium of non-inflamed tonsillar and gingival tissue biopsies. In the same biopsies, significant Ca2+ -independent citrulline-forming activity was detected. We found no difference between smoking and non-smoking subjects regarding NO-synthase expression and in vitro activity. In another group of non-smoking subjects (n=10), spraying the oropharyngeal tract with the NO-synthase inhibitor NG-monomethyl-L-arginine (250 mg) significantly reduced exhaled NO levels for at least 30 min (-18%, p<0.01). Our data suggest that cigarette smoking does not affect non-enzymatic NO formation from nitrite in saliva. However, NO is also formed by inducible NO synthase in the squamous epithelium of the normal oropharyngeal tract. We suggest that cigarette smoking may down-regulate enzymatic NO formation in the oropharyngeal compartment as well as in the bronchial compartment.

Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium.

OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the formation of PGE(2) from cyclooxygenase-derived PGH(2). Microsomal PGES-1 is induced by proinflammatory cytokines and is strongly linked to conditions that result in high PGE(2) biosynthesis. PGE(2) contributes to the pathogenesis of rheumatoid arthritis (RA), acting as a mediator of inflammation and promoting bone destruction. Induction of mPGES-1 in rheumatoid synoviocytes by proinflammatory cytokines has been demonstrated in vitro, indicating an important role in RA pathogenesis. Recent studies using mPGES-1-deficient mice demonstrated the importance of this gene in chronic inflammation. The aim of this study was to investigate the expression and localization of mPGES-1 in synovial biopsy specimens obtained from patients with RA. METHODS: Synovial tissue samples from 24 patients with RA were obtained, and immunohistologic analysis was performed using polyclonal antibodies against mPGES-1. Double immunofluorescence staining was performed with antibodies to CD3, CD19, CD20, CD68, CD163, and prolyl 4-hydroxylase. RESULTS: Intracellular mPGES-1 staining was observed in synovial membranes from all of the RA patients studied. Specifically, strong expression of mPGES-1 was detected in synovial lining cells. In sublining mononuclear and fibroblast-like cells, the extent of mPGES-1 staining was less than that in the synovial lining cells. In some patients, positive staining was observed in endothelial cells. With the double immunofluorescence technique, mPGES-1 production was detected in synovial macrophages and fibroblasts, while mPGES-1 expression was not observed in lymphocytes. CONCLUSION: The demonstration of mPGES-1 expression in synovial tissues from patients with RA suggests a role for mPGES-1 in the RA disease process. Microsomal PGES-1 might be a potential new target for treatment strategies to control PGE(2) synthesis in patients with RA, without the systemic side effects associated with cyclooxygenase inhibitors.

Inflammatory cell and cytokine patterns in patients with painful clicking and osteoarthritis in the temporomandibular joint.

The occurrence of a subset of cytokines and leukocytes in the posterior disc attachment area of the temporomandibular joint (TMJ) was investigated in two patient groups, i.e, one group with painful clicking and one with osteoarthritis. Synovial biopsies were taken during discectomy in 19 patients with painful clicking and 20 with osteoarthritis. One set of specimens was examined with immunohistochemistry, using frozen sections postfixed by para-formaldehyde and with the cell membranes permeablized in saponin. These sections were incubated with antibodies against cytokines IL-1alpha, IL-1beta, IL-1ra, TNFalpha, IFNgamma, IL2 in all patients and TGFbeta1,2,3 in 16. The other set of specimens was used to characterize cell infiltrates using immunohistochemistry with monoclonal antibodies against antigens CD68 and CD45RO, respectively. Moreover, PCNA was included as a marker for cell proliferation. The cytokine staining was most frequently positive for IL-1alpha and IL-1beta in both patient groups. However, joints with OA showed a more complex cytokine pattern, also involving IFN-gamma (P = 0.019), IL-ra (P = 0.047), and apparently but without reaching the chosen level of significance, IL-2, TNF-alpha and TGF-beta1,2,3. Positive staining for CD45RO was frequent in both groups. OA patients showed more frequently positive staining for CD68 (P = 0.025) and apparently for PCNA.

Transforming growth factor-beta (TGF-beta) and tissue transglutaminase expression in the small intestine in children with coeliac disease.

The production of cytokines from T cells and macrophages is of potential importance for the histological changes apparent in coeliac disease (CoD). Small intestinal biopsy specimens from children with CoD and disease control subjects were investigated for their content of cytokines and tissue transglutaminase (tTG). The transforming growth factor-beta1 (TGF-beta1) expression was increased in the lamina propria of children with villous atrophy. In contrast, TGF-beta3 was expressed at a higher level in the epithelium and the lamina propria of the disease control subjects. The tTG expression was increased in the small intestine of CoD patients as compared with that in subjects. Interleukin-4 (IL-4) was detected in the lamina propria of both CoD patients and controls, and some of the investigated biopsy specimens also showed IL-4 expression in the epithelium. We conclude that children with active CoD could have an altered expression of TGF-beta and tTG in the small intestine and that a disturbed regulation of TGF-beta may be of importance in the immune pathogenesis of CoD.

The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa.

BACKGROUND: Peptide antibiotics are part of the surface defences against microbial intruders. However, the presence and significance of these innate immune effectors in the skin barrier of the newborn infant have not yet been appreciated. Erythema toxicum neonatorum is an inflammatory skin reaction of unknown aetiology and significance, commonly present in the healthy newborn infant. OBJECTIVES: As peptide antibiotics are upregulated in inflammatory skin disorders, we hypothesized that this also could be the case in erythema toxicum. We also investigated if the vernix caseosa, a cream-like white substance present on the skin of the infant at birth, might contribute to host defences. METHODS: The presence of the human antibacterial peptide LL-37 was investigated by immunohistochemistry and confocal imaging of skin biopsies from four 1-day-old infants with an erythema toxicum rash and four matched newborns without the rash. In addition, we analysed the expression of LL-37 and human beta defensin-1, an antibacterial peptide of epithelial origin, by reverse transcriptase-polymerase chain reaction. Finally, we screened for antibacterial components in vernix material obtained from six healthy newborns by inhibition zone assays. RESULTS: All biopsies from the lesions of erythema toxicum showed a dense, nodular infiltrate with numerous LL-37-expressing cells located in the dermal layer and a clear localization of the peptide within CD15-expressing neutrophils, EG2-expressing eosinophils and CD1a-expressing dendritic cells. LL-37 was also found to be located in CD1a-expressing Langerhans cells and a positive staining for the peptide was seen throughout the whole epidermal layer, both in infants with and without the rash. Skin samples from infants with the rash of erythema toxicum showed a constitutive expression of human beta defensin-1, while the expression of LL-37 seemed to be induced. Furthermore, LL-37 and lysozyme were detected in the protein fractions derived from the vernix caseosa, and these fractions exhibited a clear antibacterial activity. CONCLUSIONS: Peptide antibiotics are present in the vernix caseosa and in the skin of the healthy newborn infant, indicating effective innate immune protection already during fetal and neonatal life.

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. METHODS: Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. RESULTS: Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. CONCLUSION: The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.

Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium.

OBJECTIVES: To define synovial apoptosis with respect to disease duration, inflammatory cell type, FLIP (FLICE-like inhibitory protein), and cytokines expression in patients with rheumatoid arthritis (RA). METHODS: Synovial biopsy specimens from 11 patients with longstanding RA (median disease duration 21 years) and eight with early RA (median disease duration five months) were investigated. Apoptosis (TUNEL method combined with morphological analysis), cell surface markers (CD3, CD68), cytokines (interleukin (IL) 1alpha, IL1beta, tumour necrosis factor alpha, and IL6), and FLIP expression were evaluated. Computer assisted image analysis was used for quantification. RESULTS: The apoptosis level in RA synovium was significantly higher in the group of patients with longstanding RA than in the patients with early RA (8.8% v 0.6%, p=0.001), while the number of macrophages and FLIP expression were higher in the group with early disease than in the group with longstanding RA (16.2% v 8.3%, p=0.02 and 31.1% v 0.2%, p=0.001 respectively). All three markers correlated significantly with disease duration (R=-0.7, p<0.001 for FLIP, R=0.6, p=0.001 for apoptosis, and R=-0.5, p<0.05 for CD68). Cytokine expression and T cell score were not significantly different in early RA from longstanding RA. No differences were seen between patients treated or not treated with corticosteroids or between patients treated or not treated with disease modifying antirheumatic drugs. CONCLUSIONS: The findings suggest that RA synovial macrophages are resistant to apoptosis in early RA and express high levels of FLIP. During natural or drug modified disease progression the apoptotic mechanism may be restored with a specific increase of synovial apoptosis in patients with longstanding arthritis.

Anti-tumour necrosis factor (TNF)-alpha therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis.

OBJECTIVES: Matrix metalloproteinases (MMPs) are cytokine-modulated enzymes that play an important role in the pathogenesis of rheumatoid arthritis (RA) by inducing bone resorption and cartilage destruction. This study evaluated the modulation of serum and synovial MMPs and their inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP)-1, by therapy with soluble tumour necrosis factor (TNF) alpha receptor (etanercept). METHODS: Serum samples were collected from 60 RA patients at baseline and after 8 or 12 weeks of treatment. Paired synovial biopsies were obtained from 11 patients at two time points, before and after 8 weeks of treatment. We measured serum levels of MMP-1, MMP-3 and TIMP-1 by ELISA. Immunohistological analysis of synovial tissue was performed using monoclonal antibodies specific for MMP-1, MMP-3 and TIMP-1. RESULTS: Etanercept therapy significantly down-regulated serum levels of MMP-3 and MMP-1 in parallel with the reduction in inflammatory parameters (C-reactive protein concentration and erythrocyte sedimentation rate) in RA patients. Baseline pretreatment serum levels of MMP-3 correlated with changes in clinical disease activity during therapy. No consistent changes in serum level of TIMP-1 were observed, while ratios of MMP-1 and MMP-3 to TIMP-1 were down-regulated following etanercept treatment. Immunohistochemical analyses revealed great interindividual variability, with generally a high level of expression of MMP and low expression of TIMP. No significant change in the pattern or number of positive cells occurred during therapy. CONCLUSIONS: In RA patients, etanercept therapy down-regulates serum levels of MMP-3 and MMP-1 and the ratio between MMPs and TIMP-1. This may be an important mechanism for the prevention of future development of joint damage.

Erythema toxicum neonatorum: an immunohistochemical analysis.

Erythema toxicum neonatorum is a benign rash of unknown etiology, present to various degrees in most term newborns and characterized by an accumulation of eosinophils in dermal lesions. The recruitment of leukocytes to tissues implicates the involvement of adhesion molecules, cytokines, and chemokines. We therefore performed immunohistochemistry on punch biopsy specimens from cutaneous lesions of ten 1-day-old infants with erythema toxicum using specific monoclonal antibodies directed against a variety of adhesion molecules, cytokines, chemokines, and cell type-specific membrane markers. Biopsy specimens of noninflamed skin from four matched newborns and four adults served as controls. The immunohistologic features of erythema toxicum in all 10 infants included a strong staining of the adhesion molecule E-selectin in the vessel wall and the presence of numerous inflammatory cells that were identified as dendritic cells (CD1a, CD83, HLA-DR, CD40, and ICAM-1 positive), eosinophils (EG2 positive), neutrophils (CD15 positive), macrophages (CD14, CD68, and Mac387 positive), and E-selectin-expressing cells. Furthermore, the lesions showed a high incidence of the proinflammatory cytokines interleukin (IL)-1alpha and IL-1beta and of the chemokines IL-8 and eotaxin. This immunologic activity was reduced or absent in noninflamed skin from newborn controls and adults. We conclude that there is an accumulation and activation of immune cells in the lesions of erythema toxicum, also present in noninflamed skin of 1-day-old infants, but to a lower level. The physiologic significance of the rash remains to be elucidated.

Demonstration of mast cell chemotactic activity in synovial fluid from rheumatoid patients.

OBJECTIVES: The significance of the mast cell in the pathogenesis of rheumatic diseases has become more evident. Although mast cell hyperplasia is a feature of rheumatoid arthritis, the nature of mast cell chemoattractants involved in the recruitment of mast cells in joint diseases has not been studied in any detail. In this study the presence of mast cell chemotactic activity in synovial fluids was examined. METHODS: Synovial fluids from seven rheumatoid patients were tested in a modified Boyden chamber, where a human mast cell line was used as responder. The presence of stem cell factor (SCF) and transforming growth factor beta (TGFbeta) was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: Six of the seven synovial fluids tested exhibited mast cell chemotactic activity. Two characterised human mast cell chemotaxins, SCF and TGFbeta, were highly expressed in the synovium. Soluble SCF could be detected in all fluids analysed. Blocking antibodies against SCF or TGFbeta almost completely blocked the activity in one fluid, partially blocked the activity in three, and did not affect the activity in two. Treatment of the responder cells with pertussis toxin reduced the migratory response against seven fluids, indicating the presence of chemoattractants mediating their effect through G(i) coupled receptors. CONCLUSION: These data demonstrate the presence of multiple factors in synovial fluid acting as mast cell chemoattractants, two of which are SCF and TGFbeta that contribute to the effect. These findings may be of importance for developing new strategies to inhibit mast cell accumulation in rheumatic diseases.

Systemic anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha synthesis.

OBJECTIVE: To investigate the hypothesis that tumor necrosis factor alpha (TNFalpha) blockade in rheumatoid arthritis (RA) diminishes synovial synthesis of TNFalpha, interleukin-1alpha (IL-1alpha), and IL-1beta. METHODS: Patients with active RA received a single 10 mg/kg infusion of infliximab. Multiple synovial biopsy specimens were obtained from a knee the day before infusion and 14 days later. A modified immunohistochemical method detecting cytokine-producing rather than cytokine-binding cells was applied to determine synthesis of TNFalpha, IL-1alpha, and IL-1beta in fixed, cryopreserved sections. Computerized image analysis using two different methodologies was performed by independent observers blinded to the identity of samples. RESULTS: All 8 patients met the American College of Rheumatology 20% improvement response criteria (ACR 20) at 2 weeks, and half of these patients met the ACR 50. With a few exceptions, there was concordance between both image analysis methodologies regarding the direction of change in immunopositive area fraction for all cytokines analyzed. TNFalpha synthesis was significantly reduced after treatment (P = 0.05 at the Karolinska Institute, Stockholm, Sweden; P = 0.008 at the Kennedy Institute, London, UK). Patients meeting the ACR 50 were those with the highest baseline levels of TNFalpha synthesis. There was a significant correlation between baseline levels of TNFalpha expression and change in TNFalpha levels in response to therapy. Both IL-1alpha and IL-1beta synthesis were reduced in 3 patients; IL-1alpha synthesis alone was reduced in 2 patients and IL-1beta synthesis alone was reduced in 2 patients. In 1 patient, neither IL-1alpha nor IL-1beta synthesis was reduced. CONCLUSION: Analysis of synovial tissue by means of immunomorphology and image analysis in a clinical trial setting may allow the drawing of biologically meaningful conclusions. Synovial TNFalpha synthesis was reduced 2 weeks after infliximab treatment. Reductions in IL-1alpha and IL-1beta synthesis were demonstrated in a subgroup of patients. High levels of synovial TNFalpha production prior to treatment may predict responsiveness to therapy.

An immunohistochemical analysis of cytokine expression in allergic and irritant contact dermatitis.

The purpose of this study was to investigate whether the specific and non-specific inflammatory responses to allergens and irritants give rise to immuno-histochemical detectable differences in the cytokine profile in the skin. Skin biopsies taken at 0, 6, 24 and 72 h from contact allergic reactions to nickel and from irritant reactions to sodium lauryl sulphate were analysed. The main finding was that the dermal cells expressed similar patterns of cytokines (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6 and IL-10) in both types of contact reaction at 72 h. However, two differences were observed. Staining for the IL-1 receptor antagonist was more prominent in the dermis at the late stages of the allergic reaction compared with the late stage of the irritant reaction. The other difference was an increased interferon-gamma staining of infiltrating mononuclear inflammatory cells in the dermis in the sodium lauryl sulphate group compared with the nickel group. A more rapid general onset of cytokine production was found in the sodium lauryl sulphate group than in the nickel group. The main conclusion of this study was that after 6 h the cytokine patterns did not differ between the specific and the non-specific inflammatory responses in the skin.

Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment.

OBJECTIVES: Assessment of the numbers and spatial distribution of cells producing interleukin 1alpha (IL1alpha), interleukin 1beta (IL1beta), tumour necrosis factor alpha (TNFalpha), and interleukin 6 (IL6) in the synovial membranes of patients with rheumatoid arthritis (RA). METHODS: Synovial tissue specimens from 40 patients with RA and eight patients with non-rheumatic disease were obtained by arthroscopy guided biopsy techniques or during joint surgery. A modified immunohistochemical method detecting cytokine producing rather than cytokine binding cells was applied to determine cytokine synthesis in fixed cryopreserved sections. Computerised image analysis methods provided comparative quantitative assessments. RESULTS: A wide variation between subjects was recorded for both quantities and profiles of expressed cytokines, despite similar macroscopic and histopathological features of inflammation. IL1alpha and IL1beta were the most abundant monokines identified, though produced at different sites. IL1alpha was predominantly seen in vascular endothelial cells, whereas IL1beta staining was mainly shown in macrophages and fibroblasts. Concordant results for the detection of TNFalpha at protein and mRNA levels were obtained with an unexpectedly low number of TNFalpha producing cells compared with IL1 expressing cells in many patients with RA. Specimens acquired arthroscopically from areas with maximum signs of macroscopic inflammation showed an increased number of TNFalpha producing cells in pannus tissue compared with that occurring in synovial villi of a given joint. This clustered distribution was not found for cells expressing any of the other studied cytokines. CONCLUSION: The recorded heterogeneous profile of proinflammatory cytokine synthesis in the synovial membrane among patients with RA may provide a clue for an understanding of the wide variation in responsiveness to different modes of antirheumatic treatment between patients.

Quantitative analysis of synovial membrane inflammation: a comparison between automated and conventional microscopic measurements.

OBJECTIVE: The objective of this study was to quantify selected features of chronic synovial tissue inflammation by computerised image analysis and to validate the results by comparison with conventional microscopic measurements. METHODS: Synovial biopsy samples were obtained from the knee joints of patients with chronic arthritis and prepared for immunohistochemical analysis using standard techniques. Following the development of special software, four parameters of chronic synovial inflammation were evaluated: intimal layer thickness, CD3+ cell infiltration, CD8+ cell infiltration and vascularity. Intimal layer thickness was expressed in microns. The intensity of CD3+ and CD8+ cell infiltration was expressed as the percentage area of the tissue section occupied by positively stained cells. Vascularity was expressed as the percentage area occupied by blood vessels. Conventional quantitative microscopic analysis was also undertaken and the results from both methods compared. RESULTS: Seventy eight tissue sections were selected for study. Measurements of intimal layer thickness by both techniques correlated strongly: r = 0.85, p = 0.0006. Measurements of CD8+ cell infiltration, usually widely dispersed, also correlated well: r = 0.64, p = 0.005. Measurements of CD3+ cell infiltration, often densely aggregated, correlated less well: r = 0.55, p = 0.02. Measurements of vascularity demonstrated no statistically significant correlation: r = 0.41, p = 0.07. Proficiency in the use of computerised image analysis was readily acquired. CONCLUSION: Computerised image analysis was successfully applied to the measurement of some features of synovial tissue inflammation. Further software development is required to validate measurement of blood vessels of variable size.

Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines.

The atherosclerotic lesion contains large numbers of macrophages and T lymphocytes. This suggests that a cellular immune response may take place in the lesion, and oxidized lipoproteins, heat shock proteins, and micro-organisms have been implied as candidate antigens. However, the effector mechanisms elicited by this response have been largely unclear. We have therefore analyzed endarterectomy specimens by immunohistochemistry and reverse transcription-PCR to detect immune cytokines produced by immunocompetent cells of the advanced human plaque. The pro-inflammatory T cell cytokines, interleukin-2 and interferon-7, were found in a large proportion of plaques (IL-2 in 50% and interferon-gamma in 30% of plaques by immunohistochemistry and mRNA for both cytokines in 70% of plaques by PCR). In contrast, interleukin-4 and interleukin-5 were rarely observed (both cytokines in 10% of plaques by immunohistochemistry, mRNA for interleukin-4 in 10% and for interleukin-5 in 40% by PCR). This demonstrates the presence of a predominantly pro-inflammatory, Th1-type T cell response in atherosclerosis. This conclusion was further supported by the expression of the pro-inflammatory cytokine, interleukin-1 by plaque macrophages and endothelial cells. In addition, the chemokine interleukin-8 and the macrophage differentiation-stimulating cytokine, granulocyte-monocyte colony stimulating factor, were observed in plaque tissues, suggesting that the micro-environment promotes monocyte recruitment and macrophage differentiation. Occasional eosinophils and B cells were, however observed, which is compatible with a microheterogeneity within the lesion. Finally, the anti-inflammatory and fibrogenic cytokines, transforming growth factor-beta1-3 and its carrier protein, latent TGF-beta binding protein, were found in large amounts in all plaques. Together, these results show that a pro-inflammatory, Thl type cellular immune response takes place in the atherosclerotic plaque. The balance between pro-inflammatory and anti-inflammatory cytokines may be decisive for the progression of the lesion.