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Zusammenfassung: Genetische Risikofaktoren gibt es bei allen Kollagenosen. Am wichtigsten sind sie beim systemischen Lupus erythematodes und bei der systemischen Sklerose (Sklerodermie), wie systematische Analysen der Familiengeschichten der Patienten zeigen. Beide Krankheiten sind genetisch miteinander und mit anderen Autoimmunerkrankungen verwandt. Dies zeigt auch die Molekulargenetik, da die gut validierten Risikofaktoren fast alle mehrere Krankheiten betreffen (PTPN22, STAT4, BANK1, TNFAIP3, IRF5, BLK). Viele weitere Risikofaktoren sind weniger gut untersucht. Der HLA-Komplex nimmt unter den genetischen Risikofaktoren eine besondere Stellung ein, da sich in dieser Region viele Loci mit zum Teil extrem hohem Polymorphismus befinden, die einen Beitrag zum gesamten Risiko leisten können.

Zusammenfassung: Die Diagnosestellung und Therapie der systemischen Sklerose (SSc) ist anspruchsvoll, nicht zuletzt aufgrund der Vielzahl der betroffenen Organsysteme und des chronischen und häufig schweren Verlaufs. Bisher gibt es weder allgemein zugängliche Empfehlungen für die Diagnostik der SSc zu Beginn der Erkrankung noch für die in der Regel während des langjährigen Krankheitsverlaufes notwendigen apparativen und klinischen Kontrolluntersuchungen. Aufgrund neuer Entwicklungen, z. B. in der Therapie der pulmonalen arteriellen Hypertonie, können diese Untersuchungen jedoch wesentlich dazu beitragen, rechtzeitig eine beginnende Organbeteiligung zu erkennen und zu behandeln. In der vorliegenden Arbeit werden die vom Deutschen Netzwerk für Systemische Sklerodermie (DNSS) erarbeiteten Empfehlungen zur organspezifischen klinischen und apparativen Diagnostik der SSc vorgestellt.

Seit langem ist die Assoziation der rheumatoiden Arthritis (RA) mit dem HLA-Komplex bekannt. Doch was bedeutet dies eigentlich? Es werden verschiedene Möglichkeiten einer immunologischen Interpretation in Erinnerung gerufen und eine neuere Variante vorgestellt. Hierbei handelt es sich um die Interaktion zwischen Komplexen aus Antigen und molekularen Chaperonen der HSP70-Familie einerseits und HLA-DR-β-Ketten andererseits, speziell der DRB1*0401-β-Kette, die die meisten RA-Patienten unserer Region tragen. Dieser Mechanismus könnte *0401+Personen Vorteile bei der Infektabwehr, aber eine höhere Anfälligkeit für Autoimmunerkrankungen bringen. Chaperonmaschinen sind im Gelenk von Patienten mit rheumatoider Arthritis hoch reguliert. Was tun sie dort? Da ihre Anzahl und Vielfalt groß ist, bestehen viele Möglichkeiten, die von Antigenpräsentation bis zu Immunregulation reichen. Neben dem HLA-Komplex spielt der „genetische Hintergrund“ für die Entwicklung einer Autoimmunerkrankung eine wichtige Rolle. Dies lässt sich in Familien von Patienten mit RA oder Sklerodermie zeigen, in denen ein hoher Anteil der erstgradig Verwandten ebenfalls an einer „verwandten“ Krankheit leidet. The association of rheumatoid arthritis (RA) with the HLA complex has been well established since 1978. But what does that mean? After reminding the reader of some existing immunological interpretations, a more recent variant is introduced. Antigens and molecular chaperones of the HSP70 family form complexes, which interact with HLA-DR β-chains, especially of the DRB1*0401 genotype, which is the most common among patients with RA in our region. This mechanism might bring *0401+persons an advantage in defence against microorganisms, but a disadvantage concerning autoimmunity. Chaperone machines are upregulated in synovial tissue of RA patients. As their number and variety is huge in humans, there exist many possibilities for function, reaching from antigen presentation to immune regulation. In addition to the HLA complex, the “genetic background” plays an important role for the development of an autoimmune disease. This is demonstrated in families of patients with RA or scleroderma, where a high percentage of first degree relatives suffer from a “related” disease.

Human B cell hybridomas were established to define new autoantigens of importance for autoimmune diseases such as rheumatoid arthritis (RA). One lgG1, λ monoclonal antibody (FKN-E12), was derived from synovial B lymphocytes of a patient with sero-negative RA. The purified lg was used to select specifically binding peptides from a random peptide phage display library. Only one epitope with the heptamer sequence HLTFGPG was detected and named RASFp1. Very similar and partly identical sequences are found in the variable region of lg κ light chains in position 96–101, at the junction of framework 2 and the J-region. The antibody FKN-E12 was shown to detect the epitope RASFp1 also on human lgG κ chains, but only in a specific conformation. The aim of the present study was to analyse human sera from patients with autoimmune diseases, non-autoimmune inflammatory diseases and healthy blood donors for the presence of lgG binding to RASFp1. For this purpose a 15-mer-peptide was synthesized containing RASFp1 within Vk-derived flanking regions, and an ELISA assay established. Sera of 142 individuals were studied. Only <5% of the control sera including sera from patients with non-autoimmune inflammations were positive. In contrast, 45% of sera from patients with RA or SLE contained RASFp1-binding antibodies. Within the 40 RA sera analysed so far, rheumatoid factors and RASFp1-binding antibodies have shown no correlation with each other.

To investigate a possible relationship between the presence of anti-neutrophil cytoplasm antibodies (ANCA), rheumatoid factors (RF), anti-nuclear antibodies (ANA), disease severity and HLA-DR phenotypes, 46 consecutive ANCA+ and 48 ANCA-, clinically well-documented RA patients were studied for RF, ANA and HLA-DR phenotypes. The 46 ANCA+ patients showed predominantly an atypical perinuclear staining pattern (89%). ANCA positivity was associated with higher RF titres (P<0.005) and advanced functional Steinbrocker grades III/IV (P<0.015). ANCA+ patients were also more often positive for ANA than ANCA- patients (P<0.008). There was no correlation between ANCA positivity and certain HLA-DR phenotypes although the frequency of DR4+ (67% vs 52%) and, in particular, of DR4+ blanks (phenotypically homozygous) was increased in ANCA+ as compared to ANCA- patients (20% vs 8%). DR4-DR1- RA patients were twice as frequent in the ANCA- than in the ANCA+ group (22.9% vs 8.7%). Correspondingly, the DR4+DR1- phenotype was increased among ANCA+ RA patients. Regarding functional Steinbrocker grades, the DR4+ phenotypes were slightly but not significantly increased in grades III and IV whereas ANCA positivity was significantly associated with severe functional Steinbrocker grades III/IV (66% ANCA+ vs 39% ANCA-,P<0.015). ANCA positivity identified a population of RA patients with a long-standing and severe clinical course of the disease. There was no correlation between ANCA positivity and certain HLA-DR phenotypes.

Summary: This study of 110 seropositive rheumatoid arthritis (RA) patients confirms the significant association of susceptibility to RA with HLA-DR4 specificity (P<0.001). The DR1 frequency is elevated in the entire seropositive patient group, reaching marginal significance (P<0.025). The DR4-negative patients, however, have a much higher prevalence of DR1 (P<0.001). Surprisingly, the DRw6 specificity is significantly increased in the remaining DR4- and DR1-negative patients (P<0.01). These results demonstrate that RA is not associated with a single HLA-specificity, but to various degrees with DR4, DR1, and DRw6. These findings, and particularly the newly recognized association with DRw6, support the hypothesis that functionally equivalent shared epitopes or conformations on otherwise distinct MHC molecules may confer risk for developing RA.

CD21, the complement receptor type 2 (CR2), binds the complement fragments iC3b, C3dg and C3d, interacts with CD23 (the low-affinity receptor for IgE), and binds IFN-α. This 145 kDa glycoprotein merits particular interest because it plays a pivotal role in the activation and proliferation of B cells by lowering the signal threshold. In human disease CD21 is important as a receptor for Epstein-Barr virus and HIV. CD21 is primarily expressed on B lymphocytes and follicular dendritic cells, but has also been reported on T cells. We established a semi-quantitative PCR and compared the CD21 mRNA levels of B and T lymphocytes with the expression of the CD21 glycoprotein on the surface of the respective cells by flow cytometry. The B cell lines Raji and Ramos and the T cell lines Jurkat and Molt4 expressed equal amounts of CD21 mRNA, but differed in surface staining. To address the question to which extent primary human B and T lymphocytes express CD21 mRNA and membrane-bound CD21 glycoprotein, we separated B cells, CD4⁺ and CD8⁺ T cells from peripheral blood mononuclear cells of healthy donors. B lymphocytes and CD4⁺ or CD8⁺ T cells expressed similar amounts of CD21 mRNA. Nevertheless only B cells, but not CD4⁺ or CD8⁺ T cells, expressed detectable amounts of CD21 on their cell surface. Expression of the CD21 exon 11 has been reported being restricted to follicular dendritic cells only. To the contrary, we found that both purified B and T cell subpopulations expressed CD21 mRNA with and without exon 11.

Macrophages differ from other antigen-presenting cell types in their potential to take up, process and present particulate antigens as well as proteins and peptides. To study their influence on T cell activation we transfected the mouse macrophage-like cell line P388D1 with human genes coding for the alpha and beta chains of class II molecules (DRA*0101 and DRB1*0401 or DRB1*0404) or with a series of localized DR beta mutants. The transfected cells (TP cells) all expressed DR4 molecules on their surface. Since they stimulated some, but not all, human DR4-reactive T cell clones, some of the resident peptides are evidently similar in mouse and man. Proteins and polypeptides such as pepsin or the human acetylcholine receptor (AChR) alpha 37-181 were also processed correctly by these transfectants and then presented efficiently to other T cell clones. Nearly all the mutants tested could present to the pepsin-specific clone, establishing functional expression of the transfected DR molecules. For the more exacting AChR alpha 144-156-specific T cell clone PM-A1, we confirmed that the single Gly86--> Val mutation in the DR beta chain abolished presentation by two DR4 subtypes. If, however, this same replacement was made in a third variant that has Lys71 (rather than Arg71), the effects were less drastic. This approach could also be used to analyse the contributions of individual substitutions that confer susceptibility to rheumatoid arthritis.

CD21, the complement receptor type 2 (CR2), binds the complement fragments iC3b, C3dg and C3d, interacts with CD23 (the low-affinity receptor for IgE), and binds IFN-alpha. This 145 kDa glycoprotein merits particular interest because it plays a pivotal role in the activation and proliferation of B cells by lowering the signal threshold. In human disease CD21 is important as a receptor for Epstein-Barr virus and HIV. CD21 is primarily expressed on B lymphocytes and follicular dendritic cells, but has also been reported on T cells. We established a semi-quantitative PCR and compared the CD21 mRNA levels of B and T lymphocytes with the expression of the CD21 glycoprotein on the surface of the respective cells by flow cytometry. The B cell lines Raji and Ramos and the T cell lines Jurkat and Molt4 expressed equal amounts of CD21 mRNA, but differed in surface staining. To address the question to which extent primary human B and T lymphocytes express CD21 mRNA and membrane-bound CD21 glycoprotein, we separated B cells, CD4+ and CD8+ T cells from peripheral blood mononuclear cells of healthy donors. B lymphocytes and CD4+ or CD8+ T cells expressed similar amounts of CD21 mRNA. Nevertheless only B cells, but not CD4+ or CD8+ T cells, expressed detectable amounts of CD21 on their cell surface. Expression of the CD21 exon 11 has been reported being restricted to follicular dendritic cells only. To the contrary, we found that both purified B and T cell subpopulations expressed CD21 mRNA with and without exon 11.

Aged killer (AK) T cells are antigen-independent, IL-2-requiring variants of antigen-dependent CTL clones that have lost their original antigen specificity and have acquired, instead, specific cytotoxicity for P815 target cells. In this report we study whether AK cells use a similar or a different target cell recognition system than that of bona fide CTL. To this end, we selected from a cloned AK line variants that are partially or completely deficient in specific target recognition and/or in cytotoxic function, and analyzed these variants for expression of the T cell antigen receptor and of Lyt-2. Variants were selected from the prototype AK line (Cl 96) with specific, as well as lectin-facilitated, cytotoxicity for P815 tumor cells. Variants could be grouped into four types with increasing degrees of functional deficiency, which correlated with loss of T cell receptor and/or loss of Lyt-2. In short, loss of Lyt-2 was reflected in loss of specific target recognition, and loss of the T cell antigen receptor was reflected in loss of all cytotoxic activity. We conclude from these results that both Lyt-2 and the T cell antigen receptor are required for specific target cell recognition and the T cell antigen receptor is, in addition, required for cytotoxic function. Moreover, since AK cells express a somatically acquired specificity that differs from that of their clonal precursors, it appears that cytotoxic T cells may change their antigen receptor from one specificity to another during tissue culture.

A limiting-dilution system is described that makes use of T cell growth factor T cell expansion and allows the determination of precursor frequencies for various regulatory and effector T cells in nonimmune, polyclonally, or specifically activated T cell populations. Two different sets, a frequent and a rare set, of T helper cell precursors with specificity for trinitrophenyl-group A streptococcal vaccine, could be identified: the frequent set is of the Lyt-123 phenotype, and is present at frequencies of from 1/1,000 to 1/6,000 splenic T cells. It is only active at low cell numbers, whereas it is completely inactivated at greater cell numbers, presumably by suppressor T cells of lower frequency but greater potency. The rare set is of the Lyt-1 phenotype, is present at frequencies of from 1/10,000 to 1/70,000, and is not sensitive to suppressor cells present within the tested cell numbers. We suggest that the frequent set contains primiary helper cell precursors, whereas the rare set contains helper T memory cells preselected by previous exposure to other antigens. The results are discussed with respect to other reports on the involvement of more than one set of helper cells in antibody production.

Splenic T cells exposed to concanavalin A (Con A), and subsequently to factors produced by rat spleen cells in response to Con A (Con A sup), acquire the ability to function as helper T (TH) cells in response to xenogeneic erythrocytes (RBC). Help is measured as the reconstitution of the plaque-forming cell response of a spleen cell population depleted of T cells by treatment with anti-Thy-1 serum and complement. We propose that precursor TH cells differentiate during the in vitro treatment into mature TH cells. As differentiation occurs under limiting dilution conditions, an estimation of the precursor frequency should in principle be possible. However, a single-hit Poisson distribution does not fit our data. Instead, we observe, dependent on the T cell concentration, three separate "peaks" of response. In many experiments, using sheep, horse, and chicken RBC as antigens, we reproducibly find these "peaks" at 40-190, 600-3,000, and 20,000-100,000 T cells, placed into limiting dilution cultures, respectively. By various experiments we can show that the helper activity is not due to passively transferred rat factors, but to the titrated cells themselves. The active cell is a T cell that appears to function in an antigen-specific way and to require direct cell contact to do so. It thus resembles the classical helper T cell. As we find precursor TH cells already at very low concentrations of T cells, we titrated the range between 0 and 100 T cells/well carefully. The bent shape of the titration curves does not always allow a statistically satisfying regression analysis, and we therefore cannot estimate precise precursor frequencies from every experiment. However, a common sense argument can be made that these frequencies must be on the order of 1/10-1/100 T cells. We propose that the limiting dilution curves obtained in this system most likely reflect fundamentally important cellular interactions that regulate immunological effector functions. We favor a concept of independently interacting sets of helper and suppressor T cells of various frequencies, but other models are possible.

The primary antibody response of C57BL/6 mice to the 4-hydroxy-3-nitrophenylacetyl (NP) hapten is restricted to antibody molecules expressing the NPb idiotype. This idiotype is a genetic marker for V genes in the heavy chain linkage group. In the secondary response, the frequency of NPb idiotype-positive molecules within the antibody population drops to very low values. Accordingly, isolated NP binding receptors from NP-sensitized B lymphocytes are largely devoid of this idiotype. In contrast, the NPb idiotype is expressed on the majority of the receptor fractions which we consider T-cell derived. This finding suggests that the antigen receptors of T lymphocytes may be restricted to the expression of major (germ-line encoded?) heavy chain idiotypes.

Islet cell antigen (ICA) 69 is a newly-recognized islet cell antigen to which autoantibodies have been observed in prediabetic relatives of patients with insulin-dependent-diabetes mellitus (IDDM). Here we extend the earlier analysis of ICA 69 antibodies to patients with recent-onset IDDM and to patients with other immune-mediated diseases. ICA 69 antibodies were determined by Western blot using an affinity purified recombinant fusion protein of ICA 69 and maltose binding protein. ICA 69 antibody quantities were determined as titres using a titration curve of a standard serum as reference. Mean logarithmic ICA 69 antibody titres were 3.4 (±1.4) in 99 patients with acute IDDM compared to 2.8 (±0.9) in 49 healthy blood donors (p<0.001). A higher mean ICA 69 antibody titre of 4.1 (±0.8) was observed in 16 patients with rheumatoid arthritis in comparison to acute IDDM (p<0.01) and healthy control subjects (p<0.001). The percentage of sera with ICA 69 antibody titres above the 2 SD level of normal subjects was 21% in IDDM, 31% in rheumatoid arthritis and 6% in healthy blood donors. None of the patients with autoimmune thyroid disease (n=20), inflammatory bowel disease (n=9) or multiple sclerosis (n=7) had elevated ICA 69 antibodies. In IDDM, presence of ICA 69 antibodies persisted and the titre remained the same over 18 months of follow-up. The relationship of ICA 69 antibodies to islet cell antibodies (ICA) or insulin autoantibodies (IAA) was tested. The production of ICA 69 antibodies was not associated in diabetic patients with the presence of any of the two other autoantibodies. In conclusion, this study describes ICA 69 antibodies in acute IDDM and finds them to be independent of other islet autoantibodies. In addition ICA 69 is a target of humoural autoimmunity not only in IDDM but also in rheumatoid arthritis.

Limiting dilution (LD) analyses of polyclonally activated T cells yielded results suggesting the existence of multiple paired populations of effector and suppressor precursors for a number of different T cell functions and specificities analyzed. These populations occur at graded frequencies and suppression occurs within a pair but not between pairs. In this paper, we establish the mathematical basis for the interpretation of these multi-component limiting dilution results. First, we derive equations for a number of mathematical models and identify one model that both makes biological sense and can be used to reproduce experimental data. Second, within this model, we identify parameters such as the frequency of suppressive cells and the number of suppressive cells required for suppression. The results suggest that within each paired population, suppressor precursors are 20 times more frequent that effector precursors. Furthermore, a similar but variable excess of suppressor cells is required for suppression to become effective. Together with the high frequency (1/50-1/500) of most effector T cell precursors previously reported, the results suggest that up to 40% of the T cells can become involved in suppression of an antigen-specific effector T cell population. These studies may provide exact estimates for predictions to be tested in experiments on immune regulation.

Zusammenfassung: PTPN22 620W gilt als zweitwichtigster genetischer Risikofaktor für Typ-1-Diabetes und rheumatoide Arthritis. Die Molekularbiologie des Enzyms, die geographische Verbreitung der Variante und ihre Bedeutung auch für seltenere rheumatische Erkrankungen werden beschrieben.

Zusammenfassung: Als Risiken fr die Entwicklung einer rheumatoiden Arthritis (RA) sind sowohl umweltbedingte als auch genetische Faktoren anzusehen. Die RA, als chronische Entzndung, gilt darber hinaus selbst als Risikofaktor fr die koronare Herzerkrankung (KHK), da sie – oder ihre Behandlung – eine endotheliale Dysfunktion verursachen kann. Ein Polymorphismus im Promoter der endothelialen NO-Synthase (–786C/T) fhrt bei –786CC-homozygoten Personen ebenfalls zu endothelialer Dysfunktion. Dieser Gendefekt ist vermehrt bei Patienten mit RA anzutreffen und daher als genetischer Risikofaktor fr RA anzusehen.