Peter M. Villiger

Interleukin-6 and interstitial cystitis.

We analyzed the presence of the cytokine interleukin-6 in urine samples from interstitial cystitis patients. Interleukin-6 was significantly elevated in patients with interstitial cystitis (169.29 +/- 90.81 pg./ml. versus 34.8 +/- 6.35 pg./ml. in controls). Representative urine samples were analyzed with specific antibody to interleukin-6 and greater than 80% of the biological activity was neutralized. These cytokine measurements were then compared with clinical parameters and interleukin-6 levels correlated positively with the pain scores. Studies on the potential cellular origin of interleukin-6 showed increased levels in spontaneously voided urine but not in ureteral urine, which was collected during cystoscopy, suggesting that interleukin-6 is the product of activated cells in the bladder. This finding is supported by in situ hybridization analyses, which showed that interleukin-6 messenger ribonucleic acid expressing cells are located in the interstitium and epithelial layer, and within blood vessels. The implications of these findings for interstitial cystitis pathogenesis are discussed.

Monocyte chemoattractant protein-1 (MCP-1) expression in human articular cartilage. Induction by peptide regulatory factors and differential effects of dexamethasone and retinoic acid.

Monocyte influx and activation in synovial joints are important in the pathogenesis of both degenerative and inflammatory arthropathies. In this study, we demonstrate the potential of articular cartilage to directly modulate these events. IL-1-stimulated human articular chondrocytes transcribed 0.7-kb monocyte chemoattractant protein-1 (MCP-1) mRNA. In situ hybridization of cartilage organ cultures revealed MCP-1 transcripts in chondrocytes in the superficial tangential zone within 2 h of stimulation with IL-1. Chondrocytes in deeper layers responded by 4 h and reached maximum MCP-1 mRNA levels by 8-12 h. IL-1-stimulated cartilage organ and chondrocyte monolayer cultures released functional monocyte chemotactic activity. This was neutralized by a monoclonal antibody specific for MCP-1, and was associated with the synthesis and secretion of immunoreactive 13-kD and 15-kD isoforms of MCP-1. Regulators and signal transduction pathways involved with the expression of the MCP-1 gene in chondrocytes were analyzed. Steady-state mRNA levels were increased by the known chondrocyte activators IL-1, tumor necrosis factor alpha, LPS, platelet-derived growth factor, and transforming growth factor beta. In addition, leukemia inhibitory factor induced MCP-1 gene expression and protein synthesis, identifying this cytokine as a new regulator of chondrocyte function. Dexamethasone blunted the induction of MCP-1 gene expression by IL-1 and by activators of protein kinase A as well as protein kinase C signal transduction pathways. In contrast, retinoic acid strongly increased phorbol myristate acetate-induced MCP-1 expression and potentiated the effects of IL-1 and LPS. In conclusion, chondrocytes express MCP-1 in response to factors that are present in cartilage or synovium. This provides a mechanism by which cartilage can play an active role in the initiation and progression of arthritis.

Castleman's disease in POEMS syndrome with elevated interleukin-6.

POEMS syndrome is a rare multisystem affliction known for its signs, from which it also takes its acronym name “peripheral neuropathy, organomegaly, endocrinopathy, monoclonal (M) protein, and skin lesions.” Our study chronicles the course of this syndrome in a young woman with Castlemans disease (angiofollicular lymph node hyperplasia). Cerebrospinal fluid (CSF) and serum interleukin‐6 (IL‐6) levels were abnormally elevated at various times during a %month period. The authors conclude that the plasma cell dyscrasia associated with the POEMS syndrome of this patient was Castlemans disease. Elevation of serum IL‐6 levels might contribute to the pathogenesis of the POEMS syndrome. Cancer 1992; 69:2697‐2703.

Neuropeptide Y is an inducible gene in the human immune system

This study reports on neuropeptide Y (NPY) mRNA expression in human peripheral blood mononuclear cells (PBMC) and lymphoid tissues. By reverse transcription polymerase chain reaction (RT-PCR) it is shown that activated human PBMC of normal blood donors expressed the NPY gene. The PCR products had the expected size and Northern blotting demonstrated the presence of the 0.8-kb NPY mRNA. To define the subpopulations of mononuclear cells expressing this neuropeptide, purified monocytes, B cells and T cells were stimulated with specific activators. Monocytes and in vitro matured macrophages expressed NPY mRNA in response to phorbol myristate acetate (PMA). B lymphocytes expressed NPY mRNA following stimulation with antibody to surface immunoglobulin and PMA. In order to analyze whether these cell types express NPY under physiological conditions in vivo, human bone marrow, tonsil and thymus were analyzed. In situ hybridization of bone marrow revealed a small number of cells containing high levels of NPY mRNA which was also detected in RNA extracts of human thymus and tonsil. In summary, NPY is an inducible gene in human lymphocytes and monocytes and it is expressed at sites where these cells are activated in vivo.

Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation.

This study shows that cultured human articular chondrocytes express high levels of 1.4 kb prepro‐enkephalin mRNA. Chondrocytes store met‐enkephalin intracellularly and secrete this neuropeptide in mature as well as in precursor form. Gene expression is inducible by serum factors. High levels of prepro‐enkephalin mRNA are detected in proliferating chondrocytes but not in confluent, contact‐inhibited cells. Phorbol myristate acetate and dibutyryl cyclic AMP, but not dexamethasone, increase levels of prepro‐enkephalin mRNA. Furthermore, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) upregulate gene expression, whereas retinoic acid, which inhibits chondrocyte proliferation, suppresses both basal and induced gene expression. Using in situ hybridization it is shown that only 1–3% of primary chondrocytes express prepro‐enkephalin mRNA, whereas 52 +/− 12% of subcultured cells are strongly positive. Analysis of DNA synthesis, by autoradiography of incorporated [3H]thymidine, shows that these numbers correspond to the percentage of cells in S‐phase of the cell cycle. In cultures of primary chondrocytes TGF beta promotes the formation of cartilage nodules and stimulates proliferation of adherent cells. This is associated with high levels of prepro‐enkephalin mRNA in proliferating cells but not in contact‐inhibited cells in cartilage nodules. In contrast, formation of cartilage nodules, proliferation and the expression of enkephalin are suppressed by interleukin‐1 beta. In summary, expression of prepro‐enkephalin in human articular chondrocytes is differentially controlled by cartilage regulatory factors and closely associated with cell proliferation.

Retinoic acid inhibits interleukin‐1‐induced cytokine synthesis in human monocytes

Retinoids are pluripotent morphogens whose effects on gene expression are mediated through specific intracellular receptors. They have certain anti‐inflammatory effects in vivo, the basis of which is not clearly understood. To characterize mechanisms involved with potential anti‐inflammatory actions of retinoids, we studied the effects of retinoic acid (RA) on cytokine production in human peripheral blood monocytes. RA differentially modulated the expression of interleukin‐1β (IL‐10), IL‐6, and IL‐8 mRNAs depending on the inducing stimulus. While phorbol myristate acetate‐induced IL‐lβ and IL‐8 mRNA expression was increased by RA (IL‐6 could not be induced by this pathway in monocytes), IL‐lβ‐induced expression of IL‐1β and IL‐8 was markedly reduced and IL‐6 gene expression was almost completely suppressed. Lipopolysaccharide (LPS)‐induced cytokine synthesis was only slightly reduced and this required a longer preincubation (> 72 h) of monocytes with RA. IL‐l‐induced de novo synthesis of IL‐6 protein and secretion of biologically active IL‐6 were also inhibited by RA. The inhibition pattern of RA was different from that of dexamethasone, which inhibited both IL‐1 and LPS effects. In summary, our data show that RA regulates monocyte cytokine expression selectively in response to the particular stimuli. Inhibition of IL‐lβ‐induced cytokine expression provides a mechanism that can explain some of the anti‐inflammatory effects of RA.

Regulation of interleukin-6 (IL-6) expression : evidence for a tissue-specific role of protein kinase C

The regulation of IL-6 mRNA expression was studied in human blood monocytes and in the human epidermoid carcinoma cell line HEp-2. In human monocytes phorbol-12-myristate 13-acetate (PMA) did not induce IL-6 but it increased IL-1β and IL-8 mRNA levels. Furthermore, in monocytes, protein kinase C (PKC) activation by PMA even reduced IL-1-induced IL-6 mRNA, and IL-1-induced IL-6 synthesis was increased by the PKC inhibitor staurosporine. IL-6 synthesis in HEp-2 cells was induced by IL-1, PMA, and calcium ionophore A 23187 but not by dibutyryl-cAMP. PMA-, but not IL-1-induced IL-6 synthesis in HEp-2 cells was inhibited by staurosporine. PMA pretreatment of HEp-2 cells abolished PMA-induced IL-6 but the IL-1 effect was not reduced. These data indicate that IL-6 can be induced by a PKC-independent pathway in monocytes and HEp-2 cells. In monocytes PKC activation does not induce IL-6 and PMA interferes with the IL-1 effect. Transcription factors known to be involved with the regulation of IL-6 expression were studied by gel retardation assays. NF-IL-6 and AP-1 activity were constitutively expressed in monocytes and HEp-2 cells under conditions where IL-6 mRNA was not detectable and levels did not change in response to stimulation by IL-1 or PMA. In contrast, NF-kB was increased by both IL-1 and PMA, but only the effect of PMA, and not that of IL-1, was inhibited by staurosporine. In summary, these results show tissue-specific differences in the regulation of IL-6 expression. Induction of IL-6 in monocytes is PKC independent. In the epithelial cell line HEp-2 IL-6 is inducible by PKC as well as by a PKC-independent pathway.

Increased Interleukin-6 (IL-6) Production in a Young Child with Clinical and Pathologic Features of Multicentric Castleman's Disease

A 21-month-old boy presented with a papular rash, lymphadenopathy, and splenomegaly. He developed symmetric polyarthritis, fever, and progressive glomerulonephritis. Serologies for viral agents including HIV were negative. Antinuclear antibody was transiently positive, but no anti-DNA antibodies were present. CH50 and serum C3 values were low. Biopsies of skin, kidney, bone marrow, and lymph node were obtained. There was a perivascular and periadnexal lymphocytic infiltrate in the skin, with a normal epidermis. Renal biopsy showed proliferative mesangial glomerulonephritis. Bone marrow showed an increased number of plasma cells. Lymph node showed histologic changes described in multicentric Castlemans disease including marked follicular hyperplasia, vascular proliferation, and interfollicular expansion with numerous plasma cells. IL-6 mRNA was demonstrated in cells in the marginal zone and interfollicular regions of the node byin situ hybridization. Likewise, the serum IL-6 level was elevated during a clinical exacerbation of the patients nephritis. These data suggest an underlying lymphoproliferative disorder, such as Castlemans disease, with overproduction of IL-6 resulting in systemic features of the disease, including glomerulonephritis.

Biological agents in monotherapy for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease, which results in joint destruction and permanent disability. The advent of disease-modifying antirheumatic drugs (DMARDs) has made a profound impact on the outcome and prognosis of RA. Methotrexate (MTX) is a central agent in RA therapy, and is used either alone or in combination with biological DMARDs. However, a large proportion of RA patients (20%-40%) either do not respond to or are unable to tolerate MTX or the alternative agents used in place of MTX (including leflunomide, sulfasalazine, azathioprine, hydroxycholoquine and combination DMARDs). For these patients, monotherapy with biological DMARDs is a key treatment option that balances tolerability with improved clinical outcomes. This article reviews the data for four biological agents approved for use as monotherapy in Switzerland (adalimumab, certolizumab pegol, etanercept and tocilizumab) in order to formulate a consensus statement on their roles in biologic monotherapy of RA.

Retinoic acid inhibits IL-1-induced IL-6 synthesis in epithelial cells

The regulation of interleukin-6 (IL-6) expression by IL-1 and retinoic acid (RA) was studied in the human epidermoid carcinoma cell line, HEp-2.IL-6 synthesis in HEp-2 cells was induced by IL-1, phorbol myristate acetate (PMA) and calcium ionophore, A23187, but not by dibutyryl-cAMP.RA at 10 mol/L almost completely inhibited IL-1-induced IL-6 production in HEp-2 cells and was less effective in inhibiting the effects of PMA and A23187. Maximal inhibitory effects of RA were observed after 3 days preincubation.To characterize mechanisms involved with the RA effects, we analysed the DNA binding proteins, AP-1, a known target for RA, as well as nuclear factor (NF) IL-6, and NF kB which are involved with the induction of IL-6. Nuclear extracts of HEp-2 cells showed that IL-1 increased only NF kB binding activity and did not change the levels of constitutively expressed AP-1 and NF IL-6. RA did not affect the IL-1-mediated increase in NF kB nor the constitutive levels of AP-1 and NF IL-6 under conditions where it effectively inhibited IL-1-induced IL-6 expression. This demonstrates that RA does not inhibit IL-6 induction via downregulation of IL-1 receptors or inhibition of IL-1 signal transduction pathways. RA might act by inducing other inhibitory factors or by blocking other not yet identified factors essential for IL-1-induced IL-6 synthesis.