Michael Seitz

Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes.

The responses of lymphocytes to six CC chemokines–MCP‐1, MCP‐2, MCP‐3, MIP‐1α, MIP‐1β, and RANTES–were studied using cloned human CD4+ and CD8+ T cells. All CC chemokines tested induced migration of both types of lymphocytes, whereas two CXC chemokines used as controls, IL‐8 and IP‐10, were inactive. The monocyte chemotactic proteins (MCP‐1, MCP‐2, and MCP‐3) showed a typically bimodal concentration dependence, and were considerably more effective than MIP‐1α, MIP‐10, or RANTES. All CC chemokines also induced a rapid and transient rise in cytosolic free Ca2+ in either type of T cell. The rise was prevented by Bordetella pertussis toxin treatment, indicating that G‐protein‐coupled receptors are involved in signaling. It was most pronounced with MCP‐1 and MCP‐3, which is in agreement with the efficacy of these chemokines as chemoattractants. The responses to MCP‐2, MIP‐1α, MIP‐1β, and RANTES were weaker, and no changes were obtained on stimulation with IL‐8 or IP‐10. Freshly isolated human blood lymphocytes were also tested, but neither migration nor Ca2+ changes were observed. Low numbers of high‐affinity receptors for MCP‐1 were found on CD4+ and CD8+ cells (<900 per cell, Kd <1 nM), and desensitization experiments showed that MCP‐1, MCP‐2, and MCP‐3 share receptors. Owing to their superior effectiveness on CD4+ and CD8+ T cells, the monocyte chemotactic proteins could play a major role in the recruitment of activated T lymphocytes.—Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M., Moser, B. Monocyte chemotactic proteins MCP‐1, MCP‐2, and MCP‐3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 8: 1055‐1060; 1994.

Tocilizumab for induction and maintenance of remission in giant cell arteritis: a phase 2, randomised, double-blind, placebo-controlled trial

BACKGROUND Giant cell arteritis is an immune-mediated disease of medium and large-sized arteries that affects mostly people older than 50 years of age. Treatment with glucocorticoids is the gold-standard and prevents severe vascular complications but is associated with substantial morbidity and mortality. Tocilizumab, a humanised monoclonal antibody against the interleukin-6 receptor, has been associated with rapid induction and maintenance of remission in patients with giant cell arteritis. We therefore aimed to study the efficacy and safety of tocilizumab in the first randomised clinical trial in patients with newly diagnosed or recurrent giant cell arteritis. METHODS In this single centre, phase 2, randomised, double-blind, placebo-controlled trial, we recruited patients aged 50 years and older from University Hospital Bern, Switzerland, who met the 1990 American College of Rheumatology criteria for giant cell arteritis. Patients with new-onset or relapsing disease were randomly assigned (2:1) to receive either tocilizumab (8 mg/kg) or placebo intravenously. 13 infusions were given in 4 week intervals until week 52. Both groups received oral prednisolone, starting at 1 mg/kg per day and tapered down to 0 mg according to a standard reduction scheme defined in the study protocol. Allocation to treatment groups was done using a central computerised randomisation procedure with a permuted block design and a block size of three, and concealed using central randomisation generated by the clinical trials unit. Patients, investigators, and study personnel were masked to treatment assignment. The primary outcome was the proportion of patients who achieved complete remission of disease at a prednisolone dose of 0·1 mg/kg per day at week 12. All analyses were intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01450137. RESULTS Between March 3, 2012, and Sept 9, 2014, 20 patients were randomly assigned to receive tocilizumab and prednisolone, and ten patients to receive placebo and glucocorticoid; 16 (80%) and seven (70%) patients, respectively, had new-onset giant cell arteritis. 17 (85%) of 20 patients given tocilizumab and four (40%) of ten patients given placebo reached complete remission by week 12 (risk difference 45%, 95% CI 11-79; p=0·0301). Relapse-free survival was achieved in 17 (85%) patients in the tocilizumab group and two (20%) in the placebo group by week 52 (risk difference 65%, 95% CI 36-94; p=0·0010). The mean survival-time difference to stop glucocorticoids was 12 weeks in favour of tocilizumab (95% CI 7-17; p<0·0001), leading to a cumulative prednisolone dose of 43 mg/kg in the tocilizumab group versus 110 mg/kg in the placebo group (p=0·0005) after 52 weeks. Seven (35%) patients in the tocilizumab group and five (50%) in the placebo group had serious adverse events. INTERPRETATION Our findings show, for the first time in a trial setting, the efficacy of tocilizumab in the induction and maintenance of remission in patients with giant cell arteritis. FUNDING Roche and the University of Bern.

Both interleukin‐8 receptors independently mediate chemotaxis

Neutrophil leukocytes, the target cells for interleukin‐8 and related CXC chemokines, bear high numbers of two types of IL‐8 receptors (IL‐8R1 and IL‐8R2). By cDNA transfection Jurkat cell lines were generated that stably express either IL‐8R1 or IL‐8R2 (J‐I8L8R1 and J‐IL8R2). J‐IL8R1 expressed 4,000 ± 1,000 copies of IL‐8R 1, and bound IL‐8 with high affinity (K d 1–4 nM) and GROα and NAP‐2 with low affinity (K d 200–500 nM). J‐IL8R2 expressed 17,000 ± 3,000 copies of IL‐8R2, and bound all three chemokines with high affinity. Both transfectants showed a similar degree of chemotactic migration after stimulation with IL‐8, GROa and NAP‐2. All three chemokines were equally potent as attractants of J‐IL8R2, whereas IL‐8 was 300 to 1,000‐fold more potent than GROα or NAP‐2 as attractant of J‐IL8R1. The potencies, therefore, agree with the affinities of the ligands to IL‐8R1 and IL‐8R2. Our results demonstrate that both IL‐8 receptors function independently, and mediate chemotaxis in response to IL‐8 and other CXC chemokines.

Rapid induction of remission in large vessel vasculitis by IL-6 blockade. A case series.

OBJECTIVE To evaluate the effect of IL-6 blockade using tocilizumab in inducing remission of arterial large vessel vasculitides (LVV). METHODS Five consecutive patients with giant-cell arteritis (GCA) and two with Takayasus arteritis (TA) were treated by tocilizumab infusions (8 mg/kg). Tocilizumab was given every other week for the first month and once monthly thereafter. Clinical symptoms of disease activity, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level and glucocorticoid (GC) dosage necessary to maintain remission were prospectively assessed. MR angiography was performed to monitor local inflammation. RESULTS Of the seven patients three were newly diagnosed and four showed GC resistance, i.e. GC could not be lowered to less than 7.5 mg/day. The mean follow-up time was 4.3 months (range 3-7 months). All patients achieved a rapid and complete clinical response and normalisation of the acute phase proteins. Remarkably, prednisone dosage could be reduced within 12 weeks to a mean of 2.5 mg/day (range 0-10 mg/day). No relapse and no drug-related side effects were noted. CONCLUSION Collectively the data suggest that IL-6 blockade using tocilizumab qualifies as a therapeutic option to induce rapid remission in large vessel vasculitides.

Monocyte chemoattractant protein 1 and interleukin 8 production by rheumatoid synoviocytes. Effects of anti-rheumatic drugs

Activated synoviocytes are major effector cells in the pathogenesis of rheumatoid arthritis (RA) because of their capacity to secrete a variety of inflammatory mediators. Among these mediators, the chemotactic proteins monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8) are likely to contribute to the recruitment of inflammatory cells into the arthritic joint. We examined the effects of anti-rheumatic drugs on the MCP-1 and IL-8 production by cultured RA synoviocytes exposed to pro-inflammatory agonists. Both chemotactic cytokines were quantified by specific enzyme-linked immunosorbent assays (ELISA), and found to accumulate in the culture supernatants. Although the time course of formation was similar, the yield of IL-8 was three to 10-fold higher than that of MCP-1. Non-steroidal anti-inflammatory drugs inhibited the synthesis of prostaglandins, but did not influence the production and release of both chemotactic cytokines. Of three disease-modifying drugs tested, dexamethasone and gold sodium thiomalate (GST) inhibited the production of IL-8 and MCP-1, while methotrexate (MTX) was inactive. Dexamethasone reduced the production of MCP-1 and IL-8 by 20-65% and 60-80%, respectively, whilst GST inhibited MCP-1 and IL-8 synthesis in suboptimally, but not in optimally stimulated synoviocytes. Taken together, these results show that the production of MCP-1 and IL-8 is similarly affected by anti-rheumatic drugs and that dexamethasone is the most potent inhibitor suggesting that part of the anti-rheumatic action of glucocorticoids is due to prevention of accumulation of chemotactic cytokines acting on neutrophils and monocytes.

T cell-regulated neutrophilic inflammation in autoinflammatory diseases.

Previous studies of acute generalized exanthematous pustulosis, a peculiar drug hypersensitivity reaction, suggested that CXCL8-producing T cells regulate sterile, polymorphonuclear neutrophil-rich skin inflammations. In this study, we test the hypothesis of whether CXCL8-producing T cells are present in autoinflammatory diseases like pustular psoriasis and Behçet’s disease. Immunohistochemistry of normal skin revealed few CD4+ and CD8+ T cells, few CXCL8+ cells, and no neutrophilic infiltration, whereas in acute exacerbations of atopic dermatitis, numerous CD4+ T cells but few CD8+ T cells, neutrophils, or CXCL8+ cells were detected. In contrast, a pronounced infiltration of neutrophils and of predominantly CD4+ T cells was observed in skin biopsies from pustular psoriasis, Behçet’s disease, and acute generalized exanthematous pustulosis, with infiltrating T cells strongly positive for CXCL8 and the chemokine receptor CCR6. Skin-derived T cell clones from pustular skin reactions were positive for CCR6 but negative for CCR8 and secreted high amounts of CXCL8 and GM-CSF, often together with IFN-γ and TNF-α after in vitro stimulation. Moreover, some skin-derived T cell clones from Behçet’s disease and from pustular psoriasis predominantly produced CXCL8 and GM-CSF, but failed to secrete IL-5 and IFN-γ. These cells might represent a particular subset as they differ from both Th1 as well as Th2 T cells and are associated with a unique, neutrophil-rich sterile inflammation. Our findings suggest that CXCL8/GM-CSF-producing T cells may orchestrate neutrophil-rich pathologies of chronic autoinflammatory diseases like pustular psoriasis and Behçet’s disease.

Randomized, Double-Blind Comparative Trial of Subunit and Virosomal Influenza Vaccines for Immunocompromised Patients

BACKGROUND To our knowledge, no study to date has compared the effects of a subunit influenza vaccine with those of a virosomal influenza vaccine on immunocompromised patients. METHODS A prospective, double-blind, randomized study was conducted to compare the immunogenicity and reactogenicity of subunit and virosomal influenza vaccines for adult patients who had an immunosuppressive disease or who were immunocompromised as a result of treatment. RESULTS There were 304 patients enrolled in our study: 131 with human immunodeficiency virus (HIV) infection, 47 with a chronic rheumatologic disease, 74 who underwent a renal transplant, 47 who received long-term hemodialysis, and 5 who had some other nephrologic disease. There were 151 patients who received the subunit vaccine and 153 patients who received the virosomal vaccine. A slightly higher percentage of patients from the subunit vaccine group were protected against all 3 influenza vaccine strains after being vaccinated, compared with patients from the virosomal vaccine group (41% vs. 30% of patients; P = .03). Among HIV-infected patients, the level of HIV RNA, but not the CD4 cell count, was an independent predictor of vaccine response. Among renal transplant patients, treatment with mycophenolate significantly reduced the immune response to vaccination. The 2 vaccines were comparable with regard to the frequency and severity of local and systemic reactions within 7 days after vaccination. Disease-specific scores for the activity of rheumatologic diseases did not indicate flare-ups 4-6 weeks after vaccination. CONCLUSIONS For immunosuppressed patients, the subunit vaccine was slightly more immunogenic than the virosomal vaccine. The 2 vaccines were comparable with regard to reactogenicity. Vaccine response decreased with increasing degree of immune suppression. Among HIV-infected patients, the viral load, rather than the CD4 cell count, predicted the protective immune response to the vaccine. CLINICAL TRIALS REGISTRATION NCT00783380 .

Rapid induction of remission in large vessel vasculitis by IL-6 blockade

OBJECTIVE To evaluate the effect of IL-6 blockade using tocilizumab in inducing remission of arterial large vessel vasculitides (LVV). METHODS Five consecutive patients with giant-cell arteritis (GCA) and two with Takayasus arteritis (TA) were treated by tocilizumab infusions (8 mg/kg). Tocilizumab was given every other week for the first month and once monthly thereafter. Clinical symptoms of disease activity, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level and glucocorticoid (GC) dosage necessary to maintain remission were prospectively assessed. MR angiography was performed to monitor local inflammation. RESULTS Of the seven patients three were newly diagnosed and four showed GC resistance, i.e. GC could not be lowered to less than 7.5 mg/day. The mean follow-up time was 4.3 months (range 3-7 months). All patients achieved a rapid and complete clinical response and normalisation of the acute phase proteins. Remarkably, prednisone dosage could be reduced within 12 weeks to a mean of 2.5 mg/day (range 0-10 mg/day). No relapse and no drug-related side effects were noted. CONCLUSION Collectively the data suggest that IL-6 blockade using tocilizumab qualifies as a therapeutic option to induce rapid remission in large vessel vasculitides.

Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes.

OBJECTIVE To examine the potential of methotrexate (MTX) to act as a differentiation-stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). METHODS Fluorescence-activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25-OH-cholecalciferol (1,25-OH-CCF), and granulocyte-macrophage colony-stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor a, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. RESULTS MTX alone and in combination with 1,25-OH-CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas-antigen expression. Differentiation was associated with enhanced IL-1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL-1Ra release and inhibit IL-1beta synthesis in BMMC. CONCLUSION MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down-regulation of IL-1beta. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.

Infliximab inhibits bone resorption by circulating osteoclast precursor cells in patients with Rheumatoid Arthritis and Ankylosing Spondylitis

Objective: To examine the effects of infliximab on bone resorption by osteoclast precursor cells (OCPs) in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) and to compare the results with changes in disease activity. Methods: Before and during 24 weeks of infliximab treatment, peripheral blood mononuclear cells of 9 RA and 10 AS patients were seeded onto ivory wafers and adherent cells, including OCPs, were grown in medium promoting osteoclast differentiation. Bone resorption was evaluated morphometrically and correlated to disease activity. A total of 19 healthy individuals were studied in parallel. In addition, biochemical bone markers were assessed in all patients at baseline and after 24 weeks. Results: OCPs from RA patients showed a higher bone resorption at baseline when compared to AS patients. Blocking of tumour necrosis factor (TNF)α with infliximab resulted in a strong reduction of bone resorption by OCPs in both cohorts and occured faster in RA compared to AS patients. This inhibition coincided with a reduction of clinical disease activity in both patient cohorts and with an increase of serum osteocalcin levels and a relative decrease of collagen crosslinks in RA compared to AS patients. Conclusion: These results provide an explanation on the cellular level for the anticatabolic effect of TNF neutralisation on bone. The variation in the kinetics of bone resorption by the OCPs in patients with RA and AS suggests disease-specific differences in the type or in the preactivation of OCPs.