Ulrike Seitzer

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.

Localized Wegener's granulomatosis: predominance of CD26 and IFN‐γ expression

The immune response in Wegeners granulomatosis (WG) has been characterized as a predominant, potentially pathogenic Th1‐like reaction by blood T cells and T‐cell clones from diseased tissues. To elucidate further the immunopathogenic mechanisms, this study analysed the phenotypes of inflammatory infiltrates in frozen nasal biopsies with involvement of the upper respiratory tract only (localized or ‘initial phase’ WG) and with multi‐organ involvement, including systemic vasculitis (generalized WG). The expression and production of Th1 and Th2 cytokines were examined in tissue specimens and peripheral blood mononuclear cells (PBMCs) of localized and generalized WG. The number of CD3+ T cells in inflammatory infiltrates ranged from 50 to 70%, together with approximately 30% CD14+ monocytes/macrophages. An average of 40% of T cells expressed CD26 in nasal biopsies of localized WG, compared with about 16% in specimens of generalized WG. In parallel, a higher number of interferon‐γ (IFN‐γ)‐positive cells were detected in nasal tissue of localized than in generalized WG. PBMCs from localized WG similarly exhibited higher spontaneous IFN‐γ production in contrast to generalized WG (207 vs. 3u2009pg/ml, p<0.05). Interleukin‐4 (IL‐4) mRNA was found in higher amounts in generalized than in localized WG. IL‐4 production was negligible in both disease and controls. In addition, both IL‐10 mRNA and IL‐10 protein levels of activated PBMCs from localized WG were elevated when compared with generalized disease (574 vs. 154u2009pg/ml, p<0.05) or healthy controls (574 vs. 246u2009pg/ml, p<0.05). It is conluded that in nasal tissues, mainly CD4+/CD26+ T cells as well as IFN‐γ‐positive cells may support a polarized Th1‐like immune response. Furthermore, the data suggest that this in situ immune response is already initiated and established in localized WG, accompanied by increased peripheral IFN‐γ and IL‐10 production. Copyright

Differences in CCR5 expression on peripheral blood CD4+CD28− T-cells and in granulomatous lesions between localized and generalized Wegener’s granulomatosis

Wegeners granulomatosis (WG) is an autoimmune disease characterized by granulomatous lesions and a necrotizing vasculitis. Th1-type-cells lacking CD28 are expanded independent of age and immunosuppressive therapy in WG. To address their migratory properties of CD4(+)CD28(-) T-cells we studied the expression of the inducible inflammatory Th1-type chemokine receptor CCR5 in localized WG and generalized WG. Expansion of CD4(+)CD28(-) T-cells was more prominent in generalized WG compared to localized WG. In localized WG a larger fraction of CD4(+)CD28(-) T-cells displayed CCR5 expression compared to generalized WG. CCR5 expression was also higher in granulomatous lesions in localized WG. Higher levels of CCR5 expression on CD4(+)CD28(-) T-cells in localized WG may favor stronger CCR5-mediated recruitment of this T-cell subset into granulomatous lesions in localized WG. Expansion of Th-1-type CD4(+)CD28(-)CCR5(+) effector memory T-cells might contribute to disease progression and autoreactivity, either directly, by maintaining the inflammatory response, or as a result of bystander activation.

Reduced T‐cell receptor CD3ζ‐chain protein and sustained CD3ε expression at the site of mycobacterial infection

Control of mycobacterial infection by the cellular immune system relies both on antigen‐presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T‐cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T‐cell receptor signalling, we examined the protein expression of T‐cell signal transduction molecules (CD3ζ, ZAP‐70, p59fyn, p56l ck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3ζ‐chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3ε and CD3ζ expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3ε and CD3ζ expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3ζ compared to CD3ε. Using double immunofluorescence analysis, virtually no CD3ζ expression could be detected in comparison to the CD3ε expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3ζ‐chain expression, which may be restored by cytokines. IL‐2‐enhanced ζ‐chain expression and T‐cell effector functions, defined by interferon‐γ release, in M. tuberculosis‐specific and human leucocyte antigen‐DR restricted CD4+u2003T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3ζ is essential for CD3 signalling and for eliciting T‐cell effector functions, reduced CD3ζ protein expression could result in altered signal transduction and inefficient T‐cell effector functions. Alternatively, reduced CD3ζ‐chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

The use of reverse transcription polymerase chain reaction to analyse large numbers of mRNA species from a single cell.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

Genotyping in the MHC locus: potential for defining predictive markers in sarcoidosis

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.

Generation and Characterization of Multicellular Heterospheroids Formed by Human Peripheral Blood Mononuclear Cells

A three-dimensional culture system for primary human mononuclear cells was developed, which reproducibly resulted in the formation of multicellular heterospheroids. Immunohistological characterization demonstrated not only the three-dimensional tissue-like aggregation of primary monocytes, B cells and T cells, but also the presence of macrophages and proliferating cells, indicating that a differentiation of monocytes to macrophages and an activation of cells were induced. Because of the phenotypical resemblance to granulomas the influence of an in vitro infection with mycobacteria on spheroid formation and morphology was analyzed. In comparison to control incubations, the formation of multinucleated giant cells and necrotic areas containing large numbers of mycobacteria could be observed, which resembled histological hallmarks of in situ tuberculoid granulomas. These characteristics have not been described for in vitro models of granuloma formation before and thus this new culture technique may prove to be a useful tool for analyzing aspects relevant to immunopathological processes in chronically inflamed tissues.

Properties of multinucleated giant cells in a new in vitro model for human granuloma formation.

Multinucleated giant cells (MGCs) are a key feature of granulomas. They have been studied with respect to the mechanism and regulation of their formation, but the function of these cells still remains elusive. A new method for the in vitro generation of granulomas was developed and characterized in which L3 larvae of Nippostrongylus brasiliensis, as a target for the cellular response, were co‐incubated with human mononuclear blood cells. The development of epithelioid cells and MGCs was observed and single isolated MGCs were analysed by the reverse transcriptase polymerase chain reaction method. The presence of tumour necrosis factor alpha (TNFα), interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), and inducible nitric oxide synthase (iNOS) transcripts in MGCs was demonstrated. It is proposed that MGCs in the granuloma model may in part represent an active cellular constituent involved in granuloma formation and turnover and in the destruction of the irritant.

Staining pattern of seven monoclonal anti-CD26 antibodies in leprosy: implications for the use of CD26 as a surrogate marker of a human Th1-like reaction

Abstractu2002In a previous study using the monoclonal anti-CD26 antibody MIB-DS2/7 in leprosy and other granulomatous diseases, it was shown that CD26 may be a candidate for use as an operational marker of a human Th1-like reaction. In this follow-up study, we compared seven different monoclonal anti-CD26 antibodies with respect to their staining pattern in lepromatous and tuberculoid leprosy tissues. Three distinct staining patterns became apparent in this anti-CD26 antibody panel: staining of T-lymphocytes and of connective tissue; staining of T-lymphocytes, connective tissue and macrophages; and almost no staining of T-lymphocytes but staining of connective tissue and macrophages. The two antibodies assigned to the first staining pattern, including MIB-DS2/7, were found to be most suitable for the operational discrimination between Th1-like and Th2-like reactions in leprosy. The antibodies assigned to staining patterns 2 and 3 did not allow this discrimination. Although all seven monoclonal antibodies investigated were specific for CD26, only two were found to be useful in identifying a Th1-like immune reaction in human tissue.

Comparative Study of CD26 as a Th1-Like and CD30 as a Potential Th2-Like Operational Marker in Leprosy

In the last years we have been able to establish CD26 as an operational marker for a human Th1-like reaction in various granulomatous diseases. Recently, CD30 was described as a marker for a Th2-type reaction, where CD30 is preferentially expressed and its soluble form released by human T cell clones producing Th2-type cytokines. To evaluate the possibility of CD30 as an eventual operational marker for a human Th2-like reaction in vivo, we performed immunohistological stainings on frozen sections of skin biopsies from patients with lepromatous and tuberculoid leprosy. A maximum of three to four CD30-positive cells was found per section, and there was no difference in the accumulation of CD30-positive cells between the tuberculoid and the lepromatous form of leprosy. With respect to CD26-positive cells, a high number was found in tuberculoid leprosy in contrast to a greatly reduced expression of CD26 in lepromatous leprosy. We conclude that, while CD26 was confirmed as an operational marker for a Th1-like reaction in leprosy, CD30 does not represent an operational Th2 marker in this disease.