Armin Schnabel

Cytokine profiles in Wegener's granulomatosis: Predominance of type 1 (Th1) in the granulomatous inflammation

OBJECTIVE To determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegeners granulomatosis (WG), by evaluating interferon-gamma (IFNgamma) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects. METHODS Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNgamma and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB. RESULTS Patients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNgamma (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNgamma, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNgamma. CONCLUSION Our data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.

Peripheral Blood and Granuloma CD4+CD28− T Cells Are a Major Source of Interferon-γ and Tumor Necrosis Factor-α in Wegener’s Granulomatosis

To elucidate whether the fraction of CD28− T cells within the CD4+ T-cell population is a major source of Th1-like and proinflammatory cytokine production driving Wegener’s granulomatosis (WG) granuloma formation, we analyzed the phenotype and functional characteristics of peripheral blood CD4+CD28− T cells and of T cells in granulomatous lesions of 12 patients with active WG. Surface markers and intracytoplasmic cytokine and perforin expression were assessed by flow cytometry. Cytokine secretion was measured by enzyme-linked immunosorbent assay. Immunohistological studies demonstrated interferon-γ and tumor necrosis factor-α cytokine positivity attributable to CD4+CD28− T cells in granulomatous lesions. Peripheral blood CD4+CD28− T cells expressed CD57, also found on natural killer cells, and intracytoplasmic perforin. They were generally CD25 (interleukin-2 receptor)-negative. CD18 (adhesion molecule β2-integrin) was strongly up-regulated on CD4+CD28− T cells, whereas only a minority of CD4+CD28+ T cells expressed CD18. CD4+CD28− T cells appeared as a major source of interferon-γ and tumor necrosis factor-α. In contrast, CD4+CD28+ T cells were able to produce and secrete a wider variety of cytokines including interleukin-2. One-quarter of CD4+CD28+ T cells expressed the activation marker CD25, but they lacked perforin. Thus, CD4+CD28− T cells appeared more differentiated than CD4+CD28+ T cells. They displayed Th1-like cytokine production and features suggestive of the capability of CD4+ T-cell-mediated cytotoxicity. CD4+CD28− T cells may be recruited into granulomatous lesions from the blood via CD18 interaction, and may subsequently promote monocyte accumulation and granuloma formation through their cytokine secretion in WG.

Intravenous pulse cyclophosphamide in the treatment of interstitial lung disease due to collagen vascular diseases.

OBJECTIVE Substantial toxicity limits the use of daily oral cyclophosphamide (CYC) for the treatment of interstitial lung disease (ILD) due to collagen vascular diseases. We examined whether intravenous (i.v.) pulse CYC can be substituted for daily oral therapy. METHODS Six patients with rapidly progressive ILD due to polymyositis, systemic sclerosis, systemic lupus erythematosus, or primary Sjögrens syndrome received 6-9 cycles of i.v. pulse CYC (0.5 gm/m2 of body surface area), together with an initial course of 50 mg of prednisolone, which was tapered to a maintenance dosage of 5-7.5 mg/day, and their response was measured clinically, by high-resolution computed tomography (HRCT) and by assessment of the bronchoalveolar lavage (BAL) cell profile. RESULTS All patients showed significant improvement in exercise tolerance and lung function. Elevated BAL neutrophils dropped substantially, whereas the response of BAL lymphocytes was inconsistent. Low-attenuation opacities in the HRCT regressed in 4 patients and remained unchanged in 2, but reticular infiltrates remained largely unaffected. Remission was maintained with hydroxychloroquine, azathioprine, or cyclosporin A. CONCLUSION I.v. pulse CYC proved to be an effective and well-tolerated treatment in these patients. Since it appears to target mainly the inflammatory component of the disease, it should be reserved for progressive ILD featuring indices of high inflammatory activity.

CD28 negative T cells are enriched in granulomatous lesions of the respiratory tract in Wegener's granulomatosis

BACKGROUND Lack of CD28 expression on peripheral blood CD4+ and CD8+ T cells has been reported in patients with Wegeners granulomatosis (WG), suggesting a pathogenetic role of CD28– T cells in WG. METHODS Ten patients with WG and six with sarcoidosis (disease control) were analysed. Fluorescence activated cell sorter (FACS) analysis was used to detect CD28 expression on T cells from peripheral venous blood and from bronchoalveolar lavage (BAL) fluid. T cells in biopsy specimens from granulomatous lesions of the upper respiratory tract were analysed for CD28 expression by double immunofluorescence staining. RESULTS A significantly higher fraction of CD28– T cells was found in the CD4+ and CD8+ T cell compartment in BAL fluid (65.6 (5.4)% and 76.3 (4.1)%, respectively) than in blood (13.4 (6.2)% and 42.9 (6.2)%; p<0.001) in patients with WG but not in those with acute sarcoidosis (6.7 (2.2)% and 53.4 (7.3)% in BAL fluid v4.1 (2.5)% and 52.0 (9.4)% in blood). The total number of CD4+/CD28– T cells but not of CD8+/CD28– T cells was also significantly higher in BAL fluid than in blood in patients with WG (p<0.05). Patients with WG had a significantly higher fraction of CD28– T cells in the CD4+ and CD8+ T cell compartment in BAL fluid than patients with acute sarcoidosis (65.6 (5.4)% v 6.7 (2.2)%; p<0.001; and 76.3 (4.1)% v 53.4 (7.3)%; p<0.05). The total number of CD4+/CD28– and CD8+/CD28– T cells was also significantly higher in patients with WG than in those with sarcoidosis (p<0.01). An abundance of CD28– T cells was found in granulomatous lesions by double immunofluorescence staining in patients with WG. CONCLUSIONS Our data indicate enrichment of CD28– T cells in BAL fluid and suggest recruitment of CD28– T cells into granulomatous lesions in WG. Further analysis of the phenotype and function of T cell subsets in WG is needed to better understand leucocyte homing in WG and to find new therapeutic targets.

Low-dose methotrexate in rheumatic diseases--efficacy, side effects, and risk factors for side effects.

Controlled trials and observational studies have shown low-dose methotrexate (MTX) to be a second-line agent of high potency with a favorable profile of safety and tolerability in the treatment of rheumatoid arthritis (RA). Its risk-benefit ratio in psoriatic arthritis is less well documented. Preliminary reports on its beneficial effects in other disorders, including the systemic manifestations of RA, other spondyloarthritides, and collagen vascular diseases, merit more detailed examination. Gastrointestinal intolerance and hepatic enzyme elevation are the most frequent side effects of MTX; life-threatening events such as severe hemocytopenia and MTX pneumonitis are rare and amenable to prevention by recognizing risk factors and premonitory signs. Hepatotoxicity does not appear to be a major limiting factor in RA patients over the first 2 to 3 years of MTX therapy; its impact on long-term tolerance remains to be clarified.

Gene polymorphisms of immunoregulatory cytokines and angiotensin-converting enzyme in Wegener's granulomatosis

Wegeners granulomatosis is a granulomatous and vasculitic disease of unknown origin. Gene polymorphisms are known to affect phenotypes of numerous diseases. Polymorphisms within the angiotensin-converting enzyme (ACE), transforming growth factor-β1 (TGF-β1), and interleukin-10 (IL-10) genes are suspected to modify the course of granulomatous disorders. We examined whether the genotype frequencies of the named polymorphisms differ in Wegeners granulomatosis from those in healthy controls. Thirty-nine patients with Wegeners granulomatosis were genotyped for the deletion/insertion polymorphism in intron 16 of the ACE gene, a biallelic polymorphism in codon 25 of the TGF-β1 gene and a biallelic polymorphism at position –1082 of the IL-10 gene and compared with healthy blood donors. For the ACE polymorphism no significant differences were detected neither in the allele frequencies nor in the genotype frequencies. For TGF-β1 a trend to genotype CG was found. The most interesting result was the observed, significant shift to genotype AA of the IL-10 polymorphism in Wegeners granulomatosis. IL-10 and TGF-β1, immunoregulatory cytokines capable of down-regulating T helper cell type 1 response, showed a significant shift or a trend, respectively towards genotypes associated with reduced cytokine release, leading to the hypothesis that different immunoregulatory cytokine patterns dependent on gene polymorphisms might be involved in the pathogenesis of Wegeners granulomatosis.

Inflammatory cells and cellular activation in the lower respiratory tract in Churg-Strauss syndrome

BACKGROUND To obtain insight into the mechanisms of tissue injury in lung disease due to Churg-Strauss syndrome (CSS), the bronchoalveolar lavage (BAL) cell profile and the levels in the BAL fluid of cell products released by activated eosinophils and neutrophils were assessed. METHODS Thirteen patients with active progressive CSS (n = 7) or CSS in partial remission (n = 6) underwent clinical staging and bronchoalveolar lavage. The levels of eosinophil cationic protein (ECP), myeloperoxidase (MPO), and peroxidase activity in the BAL fluid were determined and the results were compared with those of 19 patients with pulmonary active Wegener’s granulomatosis (WG) and nine control subjects. RESULTS In patients with progressive CSS the BAL cell profile was dominated by eosinophils, neutrophil elevation being the exception. The eosinophilia was associated with high ECP levels (4.39 ng/ml and 0.40 ng/ml in the two CSS groups compared with unmeasurable values in the controls). Individual patients with highly active CSS also had raised MPO levels, comparable to the levels in the most active WG patients. Peroxidase activity in the BAL fluid was 1.26 U/ml and 0.10 U/ml in the two groups of patients with CSS and 0.20 U/ml in the controls. Pulmonary disease in patients with WG was characterised by an extensive increase in MPO (0.30 ng/ml versus 0.13 ng/ml in the controls) together with high peroxidase activity in the BAL fluid (4.37 U/ml), but only a small increase in ECP levels was seen. No correlation was found between the ECP and MPO levels in patients with CSS which suggests that eosinophil and neutrophil activation vary independently of each other. CONCLUSIONS These findings suggest that, in addition to eosinophil activation, neutrophil activation is an important feature in some patients with highly active CSS. The balance of neutrophil and eosinophil involvement appears to be variable and this may be one explanation for the individually variable treatment requirements of patients with CSS.

Treatment of severe neutropenia due to Felty's syndromeor systemic lupus erythematosus with granulocyte colony-stimulating factor

OBJECTIVES To examine the efficacy and safety of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for the treatment of severe neutropenia due to Feltys syndrome (FS) or systemic lupus erythematosus (SLE). METHODS Eight patients with absolute neutrophil counts (ANC) below 1,000/microL attributable to FS (n = 4) or SLE (n = 4) were treated with rhG-CSF. The hematologic and clinical response as well as side effects were recorded. In addition, reports on the use of rhG-CSF/rhGM-CSF in FS and SLE retrieved from the English language literature were analyzed. RESULTS RhG-CSF effectively corrected neutropenia due to FS and SLE in seven of the current eight patients. In 54 of 55 FS and SLE patients retrieved from the literature, G-CSF or GM-CSF, respectively, proved to be effective at elevating the neutrophil count, which was often associated with improvement of infectious complications. The neutrophil count often declined again when growth factor treatment was stopped but generally stabilized at a level that exceeded the pretreatment count. Side effects included rare cases of thrombocytopenia, arthralgias, and development of cutaneous leukocytoclastic vasculitis. Side effects were dose dependent and resolved when treatment was discontinued. One of our own patients and 17 previously reported patients continued to benefit from long-term administration of rhG-CSF over periods of more than 40 months. CONCLUSIONS RhG-CSF is an effective and generally well-tolerated treatment for neutropenia due to FS or SLE. Exacerbation of the underlying rheumatic condition due to G-CSF appears to be rare if G-CSF is administered at the lowest dose effective at elevating the ANC above 1,000/microL.

Tolerability of methotrexate starting with 15 or 25 mg/week for rheumatoid arthritis

The objective of the present study was to assess the rate of side-effects and dose-limiting toxicity in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX) at an initial dose of 15 or 25 mg/week. One hundred and eighty-five patients with active RA were enrolled into a prospective non-blind trial over 12 months and randomized to start at a dose of 15 mg/week with subsequent increases if necessary (group A) or 25 mg/week with subsequent dose reductions according to effect (group B). With 168 patients eligible for evaluation 74% of patients in group A and 73% of patients in group B were on MTX after 12 months. Withdrawal due to side-effects amounted to 16% of patients in group A and 18% in group B, and decreases in dose due to side-effects amounted to 10% in group A and 9% in group B. The higher dose of MTX elicited a significantly higher rate of gastrointestinal side-effects (28% versus 17%, P<0.05) and a tendency towards a higher rate of liver enzyme elevations (47% versus 39%). The frequencies of other side-effects did not differ significantly between the groups. We concluded that starting MTX treatment at a dose of 25 mg/week was associated with a higher rate of minor but not major toxicity as compared with 15 mg/week. With this profile of tolerability it is possible to examine the therapeutic potential of MTX doses exceeding 15 mg/week.

Long-term tolerability of methotrexate at doses exceeding 15 mg per week in rheumatoid arthritis

The objective of this study was to examie longitudinally the tolerability of methotrexate (MTX) treatment at doses exceeding 15 mg/week in an open-label, prospective study. One hundred and eighty-five patients with rheumatoid arthritis were randomized to receive 15 mg or 25 mg MTX per week initially, and were followed over 30 months. Subsequent dose adjustments according to efficacy and tolerability resulted in levelling off of the mean dose at 18 mg/week, and the original treatment groups were combined for a longitudinal study comparing toxie events during months 1–12 and months 13–30. Withdrawals due to side-effects amounted to 17% during months 1–12 and 4% during months 13–30; dose reductions due to side-effects were 9% and 7%, respectively. The annual incidence of gastrointestinal side-effects increased from 26% to 39% (P=0.05), that of liver enzyme elevation dropped from 43% to 10% (P<0.001) and haemocytopenia remained stable at 5% and 7%. MTX pneumonitis was only observed during the first year, while airway complaints without evidence of parenchymal lung involvement increased to 10% beyond the first year. Fifty-six patients experienced 65 major infectious episodes over the 30-month period, with the respiratory tract being the most frequent site. This study showed that MTX treatment at doses exceeding 15 mg/week is tolerated over extended period of time. Major toxicity and withdrawals due to side-effects occurred predominantly during the first year of treatment and thus showed a decreasing trend over time, while minor toxic events continued throughout the study with a progressive rate of mucous membrane toxicity. MTX-treated RA appears to be a risk situation for major infection.