M. R. Daha

Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that human proximal tubular epithelial cells (PTEC) synthesize a 180-kD precursor of C3 that is secreted after proteolytic cleavage into a disulphide linked two-chain molecule as found in plasma. C3 present in culture supernatants of PTEC was functionally active, however, during the culture period there was a partial inactivation of the C3 molecule as assessed by hemolytic titration. Recombinant IL-2 enhances the rate of C3 synthesis in a dose-dependent manner reaching maximal stimulation at doses of 200-400 U/ml IL-2. Northern blot analysis demonstrated a 5.2-kb C3 mRNA species present in PTEC that was increased within 24 h of IL-2 treatment. IL-2-induced enhancement of C3 production by PTEC could be neutralized with specific antibodies to IL-2. This study demonstrates that C3 synthesis in PTEC is upregulated by IL-2, the major cytokine produced by activated T cells.

Predictive value of IgG autoantibodies against C1q for nephritis in systemic lupus erythematosus.

OBJECTIVES--Antibodies against C1q (C1qAb) have been demonstrated in the serum of patients with several immune complex diseases. Patients, particularly those with lupus nephritis, were found to have increased serum titres of IgG C1qAb in a cross-sectional analysis. In the present prospective study correlations were sought between serum titres of IgG C1qAb and clinical as well as laboratory parameters of disease activity in patients with systemic lupus erythematosus (SLE). METHODS--Titres of IgG C1qAb in the serum of 68 SLE patients were measured serially during a three year period. At the same time clinical and laboratory parameters of disease activity were assessed. RESULTS--Increased titres of IgG C1qAb were found in the serum of 56% of SLE patients during the study. Significant correlations were found between increased titres of IgG C1qAb and renal involvement. Clinical signs of renal involvement were found to be associated with significant increases of serum titres of IgG C1qAb in the six months preceding this appearance. Fifty per cent of the increases in serum titres of IgG C1qAb were followed by the development of renal involvement. Elevated serum titres of IgG C1qAb were especially related to proliferative forms of glomerulonephritis. Furthermore, significant correlations were found between serum titres of IgG C1qAb and serum levels of immune complexes, levels of complement components, and titres of antibodies to DNA. CONCLUSIONS--The results suggest that IgG C1qAb play a pathogenic role in the development of lupus nephritis and that serial measurement of serum titres of IgG C1qAb is useful in the management of SLE patients.

Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes.

The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.

The influence of tetracyclines on T cell activation

Minocycline has been shown to have an anti‐inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell‐mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose‐dependent inhibition of T cell proliferation and reduction in production of lL‐2. interferon‐gamma (IFN‐γ) and tumour necrosis faetor‐alpha (TNF‐α). Besides an inhibition of IL‐2 production, mitiocycline exerted its effect on T cell proliferation by induction of a decreased IL‐2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3‐induced intracellular Ca2+ mobilization. Taken together. the results clearly indicate that minocycline has immunomodulating effects on human T cells.

Direct induction of MHC class I, but not class II, expression on endothelial cells by cytomegalovirus infection

MHC expression on endothelial cells after cytomegalovirus infection was investigated using double antibody radioimmunoassays and immunofluorescence flow cytometry. After CMV infection there was an increase in class I and beta 2 microglobulin expression. This increase corresponds to the percentage of cells that are infected with CMV. There was no evidence that the increased class I expression was due to soluble factors produced by the infected cells. Although CMV only affects approximately 10% of the endothelial cells, class I and beta 2 microglobulin expression on the cell population exceeds that induced by an optimal dose of alpha-interferon. Class II expression is not increased after CMV infection. However, when gamma-interferon was added to the medium after CMV infection a normal increase in class II expression was found, suggesting that CMV infection does not inhibit the gamma-interferon-induced expression of these molecules.

Immunoglobulin D enhances the release of tumour necrosis factor-alpha, and interleukin-1ß as well as interleukin-1 receptor antagonist from human mononuclear cells

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper‐IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and α1‐acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α), and IL‐1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL‐1β, TNF‐α, and IL‐1ra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose‐dependent and showed a 30‐fold (TNF‐α) to almost 150‐fold (IL‐1β) increase at the highest concentration (50 mg/l), while AGP (750 μg/ml) only increased the cytokine secretion fourfold (TNF‐α) to almost 30‐fold (IL‐1β). The effect of IgD on IL‐1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5‐fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL‐1β and TNF‐α, although the effect was most apparent for TNF‐α. Apart from inducing IL‐1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF‐α, IL‐1β and IL‐1ra and thus may contribute to the pathogenesis of HIDS.

Regulation and production of IL‐8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL‐8. In the present study we have analysed the production of IL‐8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum‐free conditions, were found to produce IL‐8 to different degrees from not detectable levels up to 10.8±1.5 ng IL‐8 per 1×105 cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL‐8 of PTEC is 15.1 and 8.1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0.5 ng/ml rIL‐1α or 1000 U/ml recombinant tumour necrosis factor‐alpha (TNF‐α) to the culture media of PTEC induced an up‐regulation of IL‐8 production up to 6.3‐fold and 3.0‐fold, respectively. The up‐regulation by IL‐1α and TNF‐α was dose‐ and time‐dependent. In contrast, 500 U/ml recombinant interferon‐gamma (rIFN‐γ) down‐regulated the production of IL‐8 3.4‐fold. Northern blot analysis showed that IL‐1α and TNF‐α increased the expression of IL‐8 mRNA, whereas IFN‐γ reduced IL‐8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL‐8 in the kidney, and that IL‐8 produced in the proximal tubule can be induced by various proinflammatory cytokines.

The role of Fcγ receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals

Individuals with either a late (C5–9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti‐meningococcal defence in both LCCD persons and properdin‐deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcγRIIa (CD32) and FcγRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcγRIIa and FcγRIIIb allotypes was assessed in 15 LCCD and in 15 properdin‐deficient patients with/without previous meningococcal disease, we found the combination of FcγRIIa‐R/R131 with FcγRIIIb‐NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13·9, Fisher’s test P = 0·036). No such relation was observed in the properdin‐deficient patients. The importance of FcγRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcγRIIa‐R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0·05) less than PMN from FcγRIIa‐H/H131 donors. When properdin‐deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0·001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0·6568, P = 0·01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcγR allotypes. Properdin‐deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Cell-mediated autoimmunity in patients with Wegener's granulomatosis (WG)

Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell‐mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I‐restricted T cell responses in the context of HLA‐A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA‐A*0201 on the antigen‐processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non‐fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).

The clinical significance of allospecific antibodies against endothelial cells detected with an antibody-dependent cellular cytotoxicity assay for vascular rejection and graft loss after renal transplantation.

Serum samples of 64 consecutive patients who underwent renal transplantation in our institution were examined for the presence of antibody-dependent cellular cytotoxicity (ADCC) activity against endothelial cells (EC). From each patient serum samples were obtained immediately before transplantation and 1 week, 1 month and 1 year thereafter. The results were evaluated in the context of tests to measure donor-specific humoral immunity against lymphocytes and monocytes, and related to parameters of presensitization, graft survival, and histology. Sera from 10 patients were positive for ADCC on a panel of HLA-typed endothelial cells. In 8 patients sera were already positive before transplantation and remained positive thereafter. In 4 patients a positive crossmatch with donor T and B cells and monocytes could be observed after transplantation. In only one patient were these crossmatches positive before transplantation. A significant correlation was found between ADCC positivity and vascular rejection (P=0.015); in addition graft survival was significantly better in the ADCC negative group vs. the positive group (P=0.0004). These data demonstrate the significance of allospecific anti EC antibodies for the occurrence of vascular rejection and graft loss after renal transplantation.