Cytokine mRNA in the Joints and Draining Lymph Nodes of Rats with Adjuvant Arthritis and Effects of Cyclosporin A

TNF- and IL-1 promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-, IL-1, IL-6, IFN-, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF- and IFN- mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1 and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1 and TNF- throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-, IL-1, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF- levels were reached on d20, while IL-1 peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN- mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1 or TNF- was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN- mRNA in synovium being analogous to human rheumatoid arthritis.     

Disruption of the integrity of human peritoneal mesothelium by interleukin-1β and tumor necrosis factor-α

Inflammatory and neoplastic disease processes of the abdominal cavity are frequently associated with disruption of the integrity of the peritoneal mesothelium. In the present study, we analyzed the effects of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) on the morphology and expression of adhesion molecules of human peritoneal mesothelial cells (HPMC). Treatment of HPMC with IL-1 and TNF- resulted in a time- and dose-dependent alteration of the normal cobblestone morphology of the mesothelium with loss of polarization, cellular retraction and exposure of the submesothelial matrix. The effect was already observable after 6 h of treatment and was most pronounced at a dose of 10 ng/ml of IL-1 or TNF-. These morphological alterations were associated with a significant rearrangement of the expression of mesothelial adhesion molecules as detected by flow cytometry. IL-1 and TNF- both led to a loss of the expression of the hemidesmosomal integrin subunits 6 (P<0.01 and P<0.001) and 4 (P<0.01) and an increased expression of the integrin subunit 5 (P<0.001 and P<0.01). IL-1 furthermore upregulated the expression of the integrin subunits 1, 2 and the adhesion molecule CD44 while the latter was downregulated by TNF-. Our data indicate that IL-1 and TNF- may significantly affect disease processes of the abdominal cavity by their potential to disrupt the mesothelial basal cell-matrix adhesion and, thus, the integrity of the peritoneal mesothelial cell lining.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

Poly ADP Ribose-Polymerase Inhibitors Prevent the Upregulation of ICAM-1 and E-selectin in Response to Th1 Cytokine Stimulation

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF- and IL-1 in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1 and TNF-. Blocking PARP via GPI 6150 only affected TNF- induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1 and TNF- in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1 mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF- stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.     

Chemically modified ribozyme targeting TNF-alpha mRNA regulates TNF-alpha and IL-6 synthesis in synovial fibroblasts of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is chronic polyarthritis in which a variety of inflammatory cytokines play a role. Since tumor necrosis factor- (TNF-) is one of the most important cytokines in the pathogenesis of RA, we evaluated the feasibility of ribozymes as a therapeutic agent to control the inflammatory process of RA synovium. A hammerhead ribozyme against TNF- was chemically modified to increase nuclease resistance and added to RA fibroblastlike cell cultures without using a delivery system. The cellular uptake of fluorescent-labeled ribozyme into synovial cells was found to last at least 48 hr by confocal laser scanning microscopy. The ribozyme targeting TNF- gene inhibited both the expression of TNF- mRNA and the secretion of TNF- and IL-6. The cytotoxic effect by the ribozyme on synovial cells was negligible when determined by an alamar blue assay. Chemically modified ribozymes designed to suppress the TNF- gene may be potential as a therapeutic agent for rheumatoid arthritis.     

The Pulmonary Inflammatory Response to Experimental Fecal Peritonitis: Relative Roles of Tumor Necrosis Factor-α and Endotoxin

The roles of endotoxin (LPS) and tumor necrosis factor- (TNF-) in the causation of organ injury during sepsis are unclear. To study LPS and TNF- in the genesis of lung inflammation after cecal ligation and puncture (CLP), we used endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive mice (C3H/HeOuJ). We examined lung neutrophil sequestration, interleukin 1 (IL-1) mRNA expression, IL-1 protein expression, and injury. We also determined the expression of two C-X-C chemokine mRNAs, macrophage inflammatory protein-2 (MIP-2) and KC, in the lung to determine whether in vivo, endotoxin, or TNF- are significant modulators of MIP-2 and KC mRNA expression. After CLP, increased neutrophils sequestrated in the lungs of both strains of mice and coincided with an increase in expression of IL-1, MIP-2 and KC mRNAs, and IL-1 protein. Lung and serum TNF- were significantly increased in the C3H/HeOuJ strain but not in the C3H/HeJ strain. Histologic studies of the lung revealed similar injury in both strains. Our results suggest that bacterial factors other than endotoxin cause lung neutrophil sequestration and injury after CLP and further, that TNF- production is not a prerequisite. Our findings also suggest a potential role for local pulmonary chemokine production in the control of neutrophil sequestration after CLP.     

Enhancement by TNF-α of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice

Summary The influence of tumor-necrosis-factor- (TNF-), granulocytemacrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF- and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon / (IFN/) prevented reactivation and replication.     

Inhibition of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) secretion but not IL-6 from activated human peripheral blood monocytes by a new synthetic demethylpodophyllotoxin derivative

A newly synthesized demethylpodophyllotoxin derivative, 4-O-butanoyl-4-demethylpodophyllotoxin (BDPT) or BN58705, has recently been shown to exert a potent cytotoxic activityin vitro against a variety of drug-resistant human tumor cell lines. The effect of this agent on effector cells of the immune system, however, has not been examined. The present study investigated the effect of BDPT on the response of activated human peripheral blood derived monocytes (PBM) to secrete cytokines. Activation of PBM overnight with LPS, IFN-, or PMA resulted in secretion into the supernatant of TNF-, IL-1, IL-6, and IL-8 as assessed by ELISA. The addition of BDPT to the stimulated cultures resulted in significant inhibition of TNF- and IL-1 secretion, whereas the secretion of IL-6 and IL-8 was not affected. The selective inhibition of TNF- and IL-1 secretion by BDPT-treated PBM was observed with all three stimuli tested. The inhibitory effect mediated by BDPT was concentration dependent and was optimal at 6–20 M. Time kinetic analysis indicated that the inhibition of secretion was rapid and detected as soon as 2 hr following stimulation of the PBM and lasted for as long as 24 hr. A comparison was made between BDPT and pentoxyfilline, a xanthine-derived phosphodisterase inhibitor that was reported to inhibit TNF- and IL-1 secretion by PBM. Both BDPT and PTX showed similar time kinetics and patterns of inhibition. However, the concentration used by BDPT to achieve optimal inhibition of secretion was 10- to 20-fold less than that needed by PTX. The selective inhibition of TNF- secretion by BDPT and PTX was corroborated by inhibition of TNF- mRNA but not IL-6 mRNA as assessed by RT-PCR analysis. These studies demonstrate that the antitumor cytotoxic compound BDPT is also an immunomodulatory agent that can inhibit selectively TNF- and IL-1 secretion by PBM. Further, the low toxicity and low concentrations of BDPT needed for optimal inhibition suggest that BDPT may have potential in its therapeutic application in diseases that are mediated by TNF- and IL-1 like septic shock, inflammatory responses, and infections.     

Secretion of proinflammatory cytokines by villous chorion tissue in spontaneous abortion

Secretion of proinflammatory cytokines IL-1, TNF-, IL-6, IFN-, and IFN- by the chorion tissue (8-12 weeks) was studied in normal gestation and spontaneous abortion. The production of IL-1, TNF-, and IFN- virtually did not change in spontaneous abortion, while IFN- was not secreted in all experimental groups. The production of IL-6 increased more than 2-fold in patients with spontaneous abortion during the first trimester. These data confirm the involvement of this cytokine in the reproductive processes and in the pathogenesis of miscarriage.     

Production of tumor necrosis factors by human T cell lines infected with HTLV-1 may cause their high susceptibility to human immunodeficiency virus infection

The production of tumor necrosis factor (TNF)- and TNF- by various human hematopoietic cell lines was quantitatively examined using a highly sensitive radioimmunoassay specific to TNF- or a cytolytic assay performed with mouse L929 cells. It was found that the HTLV-1-infected T cell lines examined produced large amounts of both TNF- and TNF-. In particular, interleukin-2 (IL-2)-dependent cell lines produced large amounts of TNF-. In contrast, human cell lines not infected with HTLV-1 essentially did not produce either of the TNFs. It was also found that the high production of TNF- by HTLV-1-infected cells partially correlated to their high sensitivity to human immunodeficiency virus (HIV) infection. Treatment of MT-4 cells, one of the most HIV-sensitive HTLV-1-infected cell lines, with antibody specific to TNF- reduced their sensitivity to HIV infection.     

Distinct Tumor Necrosis Factor-α Responses in Alveolar and Peritoneal Macrophages Are Associated with Local Levels of Endotoxin

Tumor necrosis factor alpha (TNF-) responses of alveolar macrophages (AMs) and peritoneal macrophages (PMs) were studied in rats after intravenous injection of lipopolysaccharide (LPS). High levels of plasma TNF-, increased pulmonary myeloperoxidase activity, and leukopenia occurred within 2 h after LPS injection. Alveolar spaces exhibited a strict compartment property, as manifested by only slightly increased LPS and TNF- levels in alveolar lavage fluid and an unchanged capacity of AMs to produce TNF-. By contrast, the peritoneal cavity had greatly increased local LPS and TNF- levels and a diminished PMs TNF- response to LPS. The amount of LPS in the alveolar spaces was less than 0.2% of the level in peritoneal fluid. These results indicate that activation of resident macrophages is dependent on the amounts of local LPS and, in addition, suggest that resident AMs neither participate in the plasma TNF- response nor contribute to neutrophil sequestration in the lung during the early stages of endotoxemia.     

Altered cytokine production in black patients with keloids

The treatment of keloids in black patients remains a medical dilemma. Previous studies have focused on primary alterations in the metabolism of fibroblasts as the key in the etiology of this condition. Yet alterations in the production of various cytokines which may alter fibroblast responses secondarily have received little attention. Twelve black patients with clinical and histological diagnosis of keloids and eight black control volunteers were studied. Peripheral blood mononuclear-cell (PBMC) fractions from both groups were assayed for production of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), alpha-interferon (IFN-), beta-interferon (IFN-), gamma-interferon (IFN-), tumor necrosis factor-alpha (TNF-), and tumor necrosis factor-beta (TNF-). The production of IFN-, IFN-, and TNF- were markedly depressed in keloid patients compared to normal controls. However, IL-1 and IL-2 production was not significantly different between the two groups. In contradistinction, keloid patients produce greater amounts of IL-6, TNF-, and IFN-. Altered levels of immunoregulatory cytokines may play a significant role in the net increase in collagen which characterizes keloid formation.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Cellular localization of inflammatory cytokines in human glomerulonephritis

We evaluated the expression of inflammatory cytokines in renal tissues obtained from 45 patients with several types of glomerulonephritis. Immunofluorescence studies with specific antibodies to interleukin (IL)-1, IL-1, IL-6, tumour necrosis factor (TNF)-, and TNF- showed intense cytoplasmic staining in the glomeruli and interstitium. Cells positive for these cytokines were found frequently in tissue from patients with lupus nephritis (WHO Class IV) and membranoproliferative glomerulonephritis, and, to a lesser extent, in tissue from patients with mesangial proliferative glomerulonephritis, Henoch-Schönlein purpura nephritis, and minimal change nephrotic syndrome. Most of these cells were dual-stained with a monoclonal antibody to monocytes-macrophages. In situ hybridization for cytokine mRNA, combined with immunoperoxidase staining for monocytes-macrophages, detected IL-1, IL-6, and TNF- mRNA in monocytes-macrophages infiltrating the glomeruli and interstitium. Occasionally, there was weak or moderate immunostaining for IL-1, IL-6, and TNF- in the glomerular mesangial and epithelial cells, but in situ hybridization signals were rarely found in these loci. These findings suggest that infiltrating monocytes-macrophages, rather than resident glomerular cells, are the major source of inflammatory cytokines in human glomerulonephritis.     

Cytokine production in whole bloodex vivo

Interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) have been reported to contribute to the pathogenesis of many inflammatory diseases, e.g., rheumatoid arthritis. As monocytes are believed to be the primary source of these cytokines in peripheral blood, the present study was conducted to establish ranges and patterns of IL-1 and TNF- secretion. Using heparinized unseparated whole blood obtained from normal human volunteers, peripheral blood monocytes were stimulated withSal. minnesota LPS or BSA/anti-BSA immune complex-coated beads (BSA-beads). ELISAs for IL-1 and TNF- were employed to quantitate cytokine levels in blood plasma without performing arduous and time-consuming extraction procedures. Over the course of a 6 hr incubation, LPS elicited a dose-dependent increase in TNF- and IL-1 production. Preincubation of whole blood with interferon- prior to the addition of a suboptimal dose of LPS or BSA-beads resulted in a synergistic potentiation of IL-1/TNF- production. Dexamethasone, utilized in the treatment of rheumatoid arthritis, proved to be a potent inhibitor of cytokine biosynthesis in whole bloodex vivo. The measurement of cytokine biosynthesis in a relevant physiologic environment not only avoids non-specific monocyte activation, but also may increase our ability to predict clinical outcomes in rheumatoid arthritis and/or other inflammatory diseases.     

Investigations on the biology,epidemiology, pathology and control of<Emphasis Type="Italic"> Tunga penetrans</Emphasis> in Brazil: III. Cytokine levels in peripheral blood of infected humans

Tungiasis is caused by penetration of the female jigger flea, Tunga penetrans, into the skin of its host. This parasitic skin disease is almost invariably associated with intense inflammation around embedded fleas, the underlying mechanisms being unknown. A study was undertaken to determine whether the inflammatory process can be attributed to immune activation induced by a biologically active foreign body. We determined the concentrations of Th1-mediated (IFN-, TNF-) and Th2-mediated (IL-4) cytokines in the sera of patients with tungiasis. The results were compared with those of controls infected with different helminths or exposed to soil-transmitted helminths. The results show that tungiasis causes a mixed Th1 and Th2 immune response, characterized by significantly increased concentrations of the pro-inflammatory cytokines IFN- and TNF-, with a slightly increased concentration of IL-4. The preponderance of the Th1 immune response was indicated by a significantly increased TNF-/IL-4 ratio in patients with tungiasis, as compared with the control groups.     

Differential effect of Fc gamma receptor ligation on LPS-stimulated TNF-alpha secretion by hepatic,splenic, and peritoneal macrophages

Lipopolysaccharide (LPS) increases serum TNF- levels due to TNF- secretion by macrophages. The serum TNF- response to LPS was augmented 10× when FcR ligation was induced by the intravenous injection of Gig-coated erythrocytes (IgG) prior to the administration of LPS. The macrophage population responsible for the augmented TNF- secretion was determined by isolating Kupffer cells, splenic macrophages and peritoneal macrophages from mice that had been given ElgG prior to LPS and determining TNF- secretion ex vivo. The intravenous injection of ElgG augmented LPS-stimulate TNF- secretion by Kupffer cells and splenic macrophages. In contrast, LPS-stimulated TNF- secretion by peritoneal macrophages was not altered by either the intravenous or intraperitoneal injection of ElgG. In vitro phagocytosis of ElgG by isolated peritoneal macrophages also did not augment LPS-stimulated TNF- secretion. These results show that FcR ligation augments LPS-stimulated TNF- secretion by Kupffer cells and splenic macrophages but not by peritoneal macrophages. Thus, the ability of FcR ligation to influence TNF- secretion may be specific to the tissue source of the macrophages.     

Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation

Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels.Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-, IL-1 and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21.Results: TNF- a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1 and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage.Conclusions: Levels of IL-1 and IL-6, but not TNF- , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1 and IL-6, rather than TNF- , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004