M. M. Eibl

Anti-inflammatory properties of human serum IgA : induction of IL-1 receptor antagonist and FcαR (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-α) and IL-6 in human monocytes

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL‐1 and TNF‐α accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL‐1 receptor antagonist (IL‐1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL‐1 activity. The present study shows that human serum IgA induces significant IL‐1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL‐1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL‐1β release, and even inhibited LPS‐induced IL‐1β release. Induction of IL‐1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of FcαR with MoAb MY‐43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA‐induced IL‐1Ra production, suggesting that FcαR‐mediated induction of tyrosine phosphorylation is required for the IgA‐induced stimulation of IL‐1Ra release. In addition, triggering of FcαR with MoAb specifically down‐regulated TNF‐α and IL‐6 release in human monocytes activated with Hib. By the induction of IL‐1Ra and down‐regulation of the release of inflammatory cytokines such as IL‐1β, TNF‐α and IL‐6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4+ and CD8+ T lymphocytes to stimulation with anti‐TCR monoclonal antibody (the first signal) in combination with anti‐CD4 or anti‐CD8, anti‐CD2 and anti‐CD28 MoAbs (the costimulatory signals) was investigated. Both CD4+ and CD8+ T cells from the patients showed significantly reduced IL‐2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL‐2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL‐2 and IL‐4 release were impaired in CD4+ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl‐type cytokines such as IL‐2, and is expressed in both CD4+ and CD8+T cell subsets.

Antibodies protect mice against challenge with tick-borne encephalitis virus (TBEV)-infected macrophages

TBEV is a flavivirus highly pathogenic for humans. By transfer of antibodies directed to the TBEV surface glycoprotein E into mice, immune protection against subsequent inoculation with free TBEV particles could be achieved. After natural TBEV infection via the skin, however, cells of the monocyte/macrophage lineage were recently demonstrated to represent an important source of local virus replication before viraemia occurs. Whether antibodies can protect against virus challenge when contracted in the form of infected cells, however, is still unclear. In the current study, TBEV antibodies protected mice against challenge with either free virus or TBEV‐infected macrophages equally well. This observation may be of more general significance.

Antigen presentation by common variable immunodeficiency (CVID) B cells and monocytes is unimpaired.

CVID is a primary immunodeficiency syndrome comprising a heterogeneous group of patients with hypogammaglobulinaemia and defective formation of specific antibodies. Previous studies demonstrated defective T cell responsiveness to antigen in a major subgroup of patients. In the present study we investigated the capacity of peripheral blood monocytes and Epstein–Barr virus (EBV)‐transformed B cell lines from seven patients with CVID, including two patients expressing an extended MHC haplotype described to be associated with CVID, to present antigen (Tet. Tox.) to CD4+ antigen‐specific T cell lines from healthy controls. The results presented show an unimpaired capacity of peripheral blood monocytes to present antigen in all patients studied. In addition, the present study demonstrates for the first time that CVID B cells function normally as antigen‐presenting cells (APC). These findings indicate that expression of a certain MHC phenotype in CVID is not associated with a defect in the presentation of recall antigen by monocytes and B cells. Based on these studies, uptake, processing and re‐expression of recall antigen in association with MHC class II molecules on the APC surface are functional and there is no indication for structural abnormalities of the MHC class II molecules expressed by the patients studied that could be essential for their function in antigen binding and presentation.

Clinical efficacy of intravenous immunoglobulin in patients with severe inflammatory chest disease and IgG3 subclass deficiency

To investigate the efficacy of i.v. IgG treatment in pediatric patients with inflammatory lung disease, a prospective, controlled clinical trial was carried out over a 2‐year study period. Patients were enrolled on the basis of severe clinical symptomatology. After 1 year of conventional treatment, the patients received 400 mg/kg per month of an i.v. IgG product containing only trace amounts of IgG3 in addition to their regular treatment throughout the second year. Significant clinical improvement, as documented by duration of hospital stay (first year 27.8 days, second year 4.9 days), use of antibiotics (132.8 versus 30.9 days) and use of steroids (21.4 versus 0.7 days) could be observed. Data obtained on a subgroup of patients with lgG3 deficiency were analysed separately. These results indicate that patients with severe chest disease who have lgG3 deficiency will also benefit from i.v. IgG treatment. The mode of action cannot be attributed to replacement of the respective isotypes, but is probably due to the effect of i.v. IgG in preventing repeated viral infections.

Different Gm allotype amounts in human intravenous immunoglobulin (IVIG) preparations; survival of foreign Gm allotypes in immunodeficient patients

IVIG is used as standard replacement therapy in primary antibody deficiency. IVIG consists mainly of IgG. IVIG preparations were investigated with respect to Gm allotypes, which are characterized by various amino acid epitopes in the constant heavy chains of the IgG subclasses IgG1, IgG2 and IgG3. The alternative allelic Gm allotypes G1m(a) and G1m(f) of IgG1, G2m(n) and G2m(”) of IgG2 and G3m(g) and G3m(b) of IgG3 were measured by sensitive competitive ELISAs for G1m(a), G1m(f), G2m(n) and G3m(b). IgG subclass levels were quantified by radioimmunodiffusion (RID). Gm allotype quantities differed significantly in various IVIG products, with different products having half or double the amount of the different Gm allotypes. The results show the effect of the different manufacturing processes, but also indicate different physicochemical properties of Gm allotypes within the same IgG subclass. The different contents of Gm allotypes might be one reason for the variable levels of specific antibodies found in IVIG products. Immunodeficient patients with homozygous expression of Gm allotypes from IGHCG1, IGHCG2 and IGHCG3 were tested after infusion of foreign Gm allotypes. A prolonged survival was found for the G2m allotype, G2m(n), compared with G1m allotypes. Different half‐lives were found for the alternative G1m(a) and G1m(f) allotypes, within the same IgG1 subclass.

Induction of anti‐mycobacterial and anti‐listerial activity of human monocytes requires different activation signals

The requirements Tor activation of anti‐mycobacterial and anti‐listerial activity of human monocytes were investigated, Human monocytes could be activated to display enhanced anti‐mycobacterial activity by a 24‐h treatment with Hpopolysaccharide, The mediator induced by this treatment was identified as being tumour necrosis factor‐alpha (TNF‐αAQl), Addition of recombinant TNF‐α (rTNF‐α) to the cultures of human monocytes for 24 h yielded comparable results (minimal dose required for induction of anti‐mycobacterial activity, 10 U/ml) Addition of anti‐TNF‐α antibody completely abrogated the effect, A similar treatment protocol failed to activate enhanced anti‐listerial activity, To trigger anti‐listerial activity, sequential treatment of human monocytcs with rTNF‐α and IL‐2 was required, Treatment of monocytes with 10 U/ml rTNF‐α for 24 h followed by incubation in the presence of 200 U/ml of IL‐2 for an additional 24 h yielded a reduction of listcrial growth which was moderate but statistically significant (P < 0.00l), The activation of monocylcs observed with rTNF‐a/IL‐2 treatment was (i) dependent on both cytokincs; (ii) sequence dependent (i,e, when IL‐2 was added prior to rTNF‐α, no effect was observed); and (iii) absent in cells treated with one cytokine only, Enhancement of anti‐listerial activity by sequential use of cytokincs was not accompanied by an increase in oxidativc burst, which indicated that oxidative mechanisms were not the reason for the observed Listeria monocytogenes growth restriction, Further support for this hypothesis was obtained after intcrfcron‐gamma treatment of human monocytcs which led to an augmented PMA‐inducible release of active oxygen radicals, but was not paralleled by growth restriction of L, monocytogenes, Our results indicate that TNI‐α plays a crucial role in the activation of monocylcs for growth restriction of intraccllular microbes, Activation of human monocytcs to restrict the growth of the facultative intracellular bacteria Mycohacterium avium intracellulare and L, monocytogenes, however, follows different patterns, the initial trigger in both cases being provided by TNF‐α‐indueed signals

Reduced IL-2 expression upon antigen stimulation is accompanied by deficient IL-9 gene expression in T cells of patients with CVID.

Patients with common variable immunodeficiency (CVID) are heterogeneous in the clinical manifesta tion of the disease as well as in the underlying mechanisms leading to the immunodeficiency. In a previous study we identified a subgroup of patients with a primary immunodeficiency disease affecting IL‐2 and IFN‐γ gene expression. The T cells of these patients revealed impaired proliferative response and reduced levels of IL‐2 and IFN‐γ ‐specific mRNA after antigen stimulation in vitro, while cellular and molecular response to phorbol ester and the calcium ionophore ionomycin (PMA + IM) or anti CD3 monoclonal antibodies (MoAbs) (OKT3) were comparable to those of healthy control individuals. Here we show that stimulation of these patients T cells with tetanus toxoid (TT) resulted in dramatically reduced levels of IL‐2, IL‐9 and IFN‐γ mRNA, while IL‐3 gene expression in three patients was comparable or even increased lo the healthy controls. As expected, addition of exogeneous IL‐2 to tetanus toxoid pulsed cultures had virtually no effect on IL‐2 transcription, but corrected the defect in IL‐9 gene expression, while IFN‐γ mRNA levels were still reduced. In conclusion, these data suggest that recombinant IL‐2 alone is not able to induce the IL‐9 gene adequately in our patients, but clearly increases IL‐9 mRNA levels in combination with tetanus toxoid.

T cell differentiation and generation of the antigen‐specific T cell repertoire in man: observations in MHC class II deficiency

The circulating T cell pool of an MHC class II‐deficient patient was shown to lack the MHC class II‐specific T cell functions. This was demonstrated by the absence of MHC class Il‐specific alloreactive T cells and a substantially decreased number of circulating CD4+ lymphocytes. The patients T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II‐deficient mice, which also lack interaction of T cell precursors with MHC class Il‐bearing accessory cells during thymic differentiation. Our data support the concept that Tcell differentiation in humans is similar, and that the human MHC‐restricted Tcell repertoire depends on prior interaction of T cell precursors with self MHC.