Polymorphonuclear leucocyte fluorescence and cryoglobulin phagocytosis in systemic lupus erythematosus.

Peripheral blood polymorphonuclear leucocytes (PMN) and cryoglobulins were isolated from patients with systemic lupus erythematosus (SLE). Fluorescent inclusions were found in PMN. Normal donor PMN were incubated with the sera and cryoglobulins from SLE patients. In most cases inclusions were observed after incubation. The high incidence of anti-IgG activity in phagocytosed cryoglobulins confirms the importance of the rheumatoid factor in phagocytosis of immune complexes. It is concluded that phagocytosis of cryoglobulins supports the suggestion that cryoglobulins are a subpopulation of immune complexes.     

Correlation of disease activity and Clq-binding immune complexes with the neutrophil inclusions which form in the presence of SLE sera

Intracytoplasmic inclusions containing immunoglobulin (Ig) and complement (C3) are found in normal neutrophils (PMN) after incubation with sera from patients with SLE. These inclusions are believed to be immune complexes removed by phagocytosis from the SLE patients' sera in vitro. Similar inclusions were also noted in the circulating PMN from some patients with SLE. In the present study we have examined the relationship between the presence of intracytoplasmic inclusions and various clinical and laboratory features of SLE. Blood from forty-five patients with SLE was drawn and separated at 37°C. Fresh heparinized blood was also obtained from normal volunteers and allowed to stand for 90 min at 37°C. The buffy coat cells from both normal and SLE groups were removed, centrifuged, washed and examined (direct method) or incubated in the SLE sera for 90 min at 37°C (indirect method). Slides of washed cells were prepared in the cytocentrifuge, stained with fluorescein-conjugated goat anti-human IgG, IgM, IgA and C3 and examined under ultra-violet light.

By the direct method, 24% of patients had small intracytoplasmic inclusions in their neutrophils when stained for IgG suggesting that immune complexes were phagocytosed in vivo. None of twenty-one normal controls had similar inclusions. By the indirect method, 62% of SLE patients were positive for IgG, 15% for IgM, 8% for IgA and 31% for C3. None of the twelve normal controls were positive.

By the indirect method, PMN inclusions containing both IgG and IgM correlated with clinical activity (P<0·001), depressed serum complement (CH50, P=0·026; and C3, P<0·051), cryoglobulinaemia (P=0·014), anti-nDNA antibodies (P<0·001) and Clq-binding immune complexes (P=0·008). A suggestive correlation with granulocytopenia was also observed. The presence of inclusions containing IgG alone did not correlate with any of these parameters. C3 and IgM appeared to be mutually exclusive, i.e. neither was present simultaneously. These findings suggest (1) that normal PMN on exposure to SLE sera develop intracytoplasmic inclusions by phagocytosis of immune complexes, (2) the presence of such complexes correlates with a number of parameters of disease activity, particularly when IgG and IgM are both present and (3) such complexes may be phagocytosed in vivo as suggested by the presence of inclusions in vivo and contribute to a number of granulocyte disturbances seen in patients with SLE. These abnormalities in granulocyte function may be important, predisposing factors for infection in patients with active SLE.

     

Immune complex detection by immunofluorescence on peripheral blood polymorphonuclear leucocytes

Peripheral blood polymorphonuclear leucocytes (PMN) from patients with SLE in active stage were isolated and frozen in gelatin capsules. Sections cut from these frozen cell preparations were examined for the presence of inclusion bodies using the immunofluorescence technique. It was demonstrated that up to 80% of the polymorphonuclear cells contain small granular inclusions when tested with anti-IgG, -IgM, -IgA and antisera specific for several C components. Incidentally large homogeneous inclusions similar to the in vitro LE cell phenomenon were observed. PMN from normal controls matched for age, sex and race showed no inclusion bodies. Three out of six patients with paraproteinaemia of different Ig class had PMN inclusions consisting of the corresponding paraprotein only. These data suggest that inclusions in PMN from SLE patients are phagocytozed immune complexes.     

Enhanced phagocytosis of immune complexes in pregnancy.

Utilizing an in vitro immunofluorescence phagocytosis assay for the detection of circulating immune complexes sera from pregnant women, from women taking contraceptive agents and age-matched controls were tested for their effect on the phagocytosis of immune complexes by normal peripheral blood polymorphonuclear leucocytes (PMN). Immune complexes present in the serum of a patient with seropositive rheumatoid arthritis and heat-aggregated IgG (COHN fraction II) were used as substrate. A significant enhancement of the phagocytosis of immune complexes by PMN in the presence of increasing amounts of pregnancy serum was seen using rheumatoid arthritis serum as well as heat-aggregated IgG. Serum from patients taking contraceptive drugs and controls showed no effect on the phagocytosis of such complexes in vitro. These observations suggest that pregnancy serum contains factors which enhance the phagocytosis of immune complexes possibly resulting in an increased clearance of such complexes. The commonly seen improvement of rheumatic diseases in the course of pregnancy might at least partially be due to this mechanism.     

Differential generation of chemiluminescence — detectable oxygen radicals by normal polymorphonuclear leukocytes challenged with sera from systemic lupus erythematosus and rheumatoid arthritis patients

Summary The generation of chemiluminescence (CL)-detectable oxygen radicals by normal human polymorphonuclear leukocytes (PMN) after challenging with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera is described. CL was measured in a luminol-dependent assay and referred to a standard obtained when preformed immune complexes (Ic) (human tetanus toxoid-antitoxoid Ic resuspended in normal pooled serum) were tested on PMN. Normal sera gave rise to CL activity by PMN between 0% and 50% of the standard Ic (mean±standard error of the mean (SEM): 20.7±4.8). Sera from SLE and RA patients induced strikingly different biological effects on PMN. SLE sera generally induced a high CL-detectable generation of oxygen metabolites which may be causally related to the intense tissue damage (vasculitis) frequently observed in this disease. In contrast to SLE, RA sera induced a CL-detectable respiratory burst by PMN that was included in the normal range. Thus, the biological effects of these sera in terms of stimulation of toxic oxygen radical generation by phagocytes are quite different. This generation of oxygen radicals might reflect a different clearance of circulating Ic by PMN in SLE and RA disease.Abbreviations CL Chemiluminescence - PMN Polymorphonuclear Leukocytes - SLE Systemic Lupus Erythematosus - RA Rheumatoid Arthritis - Ic immune complexes - TAT tetanus-antitetanus toxoid - TAT-S tetanus-antitetanus toxoid resuspended in pooled, non-inactivated human serum - SEM standard error for the mean - EHM Eagle-Hepes-Medium - PBS phosphate-buffered-saline - RF rheumatoid factor - SPA staphylococcal protein A - ANA antinuclear antibodies - KD kilodaltons - Ag antigen     

Immunological aspects of cryoprecipitates from the sera of chronic HBsAg carriers.

The sera of chronic hepatitis B surface antigen (HBsAg) carriers and seropositive controls were examined for the presence of immune complexes by cryoprecipitation. Cryoprecipitates (CP) were tested for HBsAg, antibody to HBsAg (anti-HBs), major classes of immunoglobulins, components of the complement system, rheumatoid factor and the ability to activate the alternative pathway of the complement system. For this analysis the methods employed included: radioimmunoassay, reverse passive haemagglutination, immunofluorescence, sucrose density gradient ultracentrifugation, agar-gel diffusion, immunoelectrophoresis, counterimmunoelectrophoresis, latex agglutination, and a haemolytic method for the detection of the activation of the alternative pathway of the complement system. HBsAg was frequently observed in the CP from chronic HBsAg carriers. No anti-HBs activity was detected in the serum of chronic HBsAg carriers. However, the CP from a number of chronic HBsAg carriers contained immunoglobulins and components of the complement system in the absence of rheumatoid factor, anti-HBs activity and were able to activate the alternative pathway of the complement system. On immunoelectrophoresis, a component of the CP reacting with anti-IgG, ANTI-IgA and anti-HBs antisera and demonstrating an altered (faster) electrophoretic mobility was observed. The nature of the CP strongly suggests the presence of circulating immune complexes in asymptomatic chronic HBaAg carriers. These immune complexes may be important in the eventual expression and outcome of clinical disease in apparently healthy carriers of HBsAg.     

Demonstration of circulating immune complexes by the indirect leucocyte phagocytosis test in chronic inflammatory bowel disease. Relation to results of a standard complement consumption assay

Circulating immune complexes were studied in untreated Crohn's disease (CD) and ulcerative colitis (UC) by leucocyte phagocytosis. Neutrophils from normal donors took up large immunoglobulin-containing inclusions from 14 of 15 CD sera, 3 of 15 UC sera (p less than 0.002) and from none of 15 reference sera from healthy volunteers (p less than 0.002). In contrast, inclusions could not be demonstrated on direct microscopic investigation. Our study confirms the presence of circulating immune complexes in Crohn's disease. Predominance of IgG-containing complexes in this condition is consistent with a mucosal origin. Discrepant results obtained by direct examination and by incubation of sera from patients with normal test neutrophils suggest a defective immune complex phagocytosis in CD. In consistency with this possibility, control experiments revealed a markedly decreased complex uptake by neutrophils of CD patients in vitro.     

Characterization of antigen moiety of HBsAg in the complement fixing immune complexes of hepatitis B virus positive chronic active hepatitis.

Circulating immune complexes (CIC) are frequently found in hepatitis B virus-induced chronic active hepatitis. Since antigen and antibody moieties of complexes are critical in determining many of its pathogenic factors, the constituents of these complexes were investigated with particular attention to the quantity and nature of the HBs antigen moiety of the complexes. Complement fixing immune complexes were isolated from sera of 14 patients with chronic active hepatitis by utilizing conglutinin's unique property to bind C3-fixed complexes. Low pH (2.6) was used to dissociate the complexes. Components of dissociated complexes were separated into antigen and antibody fractions using immobilized Protein A. Both fractions were analysed by electrophoresis in polyacrylamide gel with SDS. The antibody fractions showed heavy and light chains of IgG and IgM. The antigen fractions demonstrated six to 10 protein bands with mol. wt ranging between 17,000 and 120,000 daltons. To define precisely the polypeptide antigen moiety involved in the immune complex formation, a transfer blotting technique was used employing human anti-HBs globulin as probe. Polypeptides with mol. wt 97,000 and 49,000 reacted as antigen moieties of HBsAg. In addition, the levels of HBsAg in the antigen fractions were significantly greater (P less than 0.005) compared to sera from patients with acute hepatitis. Implications of these findings are discussed.     

Innate immune mechanisms in the pathogenesis of systemic lupus erythematosus (SLE).

Immune imbalance in SLE increases the susceptibility to infectious diseases. The aim of this study was to analyze several mechanisms related to non-specific immunity in this autoimmune disorder. We studied in vivo CD11b expression, phagocytosis, and chemotaxis in polymorphonuclear cells (PMN) from SLE patients. All tests were also performed under hrIL-8 stimulating conditions and analyzed by flow cytometry. Intracellular leucocyte (monocytes and PMN) enzyme activity was evaluated using specific substrates for cathepsin B and D, collagenase, and oxidative burst by flow cytoenzymology. An exaggerated in vivo CD11b expression was observed on PMN from SLE patients without noticeably in vitro effect upon hrIL-8. Similarly both, phagocytosis and chemotaxis were diminished and showed no response to hrIL-8 stimulation. The opposite was found in PMN from controls. Intracellular enzyme activity was comparable between groups as far as cathepsin B and D are concerned. A tendency of decreased oxidative-burst induction was noted in monocytes and PMN from SLE patients, whereas collagenase activity was found clearly increased in both leucocyte subpopulations. Our results may represent a deficient ability of the innate immune mechanisms for the clearance of infectious agents, immune complexes, satisfactory resolution of inflammatory processes and tissue repair in SLE.     

Immunization against hepatitis B virus by mucosal administration of antigen-antibody complexes

Antigen-antibody complexes have been shown to enhance immune responses against several antigens given by parenteral immunization. Herein, we have evaluated the potential of administering such immunostimulatory complexes by a mucosal route. Hepatitis B surface antigen (HBsAg) complexed with antibodies against HBsAg (anti-HBs) (HBsAg/Ab) was administered to BALB/c mice by intranasal inhalation. HBsAg by itself did not induce immune responses, whereas with HBsAg/Ab complexes, both systemic and mucosal immune responses were observed and these could be modulated by adjuvants. With HBsAg/Ab (1 or 10 microg), anti-HBs antibodies induced were predominantly of the IgG1 isotype (Th2-like). In contrast, anti-HBs induced by HBsAg/Ab plus cholera toxin (CT) or oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG) (1 microg each) were predominantly IgG2a (Th1-like). Results from this study indicate that HBsAg/Ab complexes can induce strong humoral immune responses when delivered by a noninvasive route, whether used alone or in combination with other mucosal adjuvants.     

Demonstration of HBsAg as the antigen component in circulating immune complexes detected by peg-solid phase test

The development of an enzyme-linked immunosorbent assay to identify HBsAg as the antigen component within circulating immune complexes using immobilized polyethylene glycol (PEG) is described. The method utilizes, on one hand, the ability of PEG to bind stably to plastic supports and, on the other, to precipitate circulating macromolecules. This method is easily performed, very cheap, quick and, above all, it helps define the biological nature of the immune complexes. HBsAg can be revealed as the antigen component of HBsAg/anti-HBs soluble immune complexes at concentrations of at least 20 ng/ml and either in antigen or antibody excess. Our results indicate that HBsAg circulates in a complexed form in 47% of HBsAg chronic carriers and in 10.7% of patients with liver disease who are positive for serum antibody to hepatitis B surface antigen (anti-HBs) and to core antigen (anti-HBc). None of the other groups of patients in the study had circulating HBsAg in the complexed form.     

Prognostic value of HBsAg/IgM complexes in hepatitis B patients: nature of the proteins involved

Hepatitis B surface antigen (HBsAg)/IgM complexes were measured using a solid phase radioimmunoassay in sera of patients with acute or chronic hepatitis B. These complexes were found in 6 out of 18 patients four weeks after the onset of the disease and only one of them developed chronic hepatitis. HBsAg/IgM complexes correlated with HBsAg, hepatitis B e antigen (HBeAg), and pre-S2 concentration. The precipitation of HBsAg/IgM reactivity by polyethylene-glycol (PEG) and the binding of this activity to the surface of certain uncoated enzyme linked immunosorbent assay (ELISA) plates indicates that HBsAg/IgM positivity may reflect the presence of circulating complexes in serum. HBsAg and pre-S2 were found as components of the complexes but anti-HBs, anti-pre-S2, and polymerized human serum albumin (pHSA) were not. An immune binding between HBsAg and IgM is still questionable. Whatever the nature of the HBsAg/IgM complexes, their detection does not seem to be an earlier indicator of prognosis than HBeAg and/or pre-S2.     

Immune complexes in sera of children with HBV-mediated glomerulonephritis

Multiple serum samples from 27 children with hepatitis B virus (HBV)--mediated glomerulonephritis (GN) were screened for the presence of immune complexes by an antigen-specific method. For this purpose an immunoenzymatic test was set up, applying a solid-phase Clq and the enzyme-conjugated antibodies: anti-HBs and anti-HBe. Complexes of HBsAg were found in sera of 15 children (55.6%), while complexes of HBeAg--in sera of 12 children (44.4%). Molecular weight of complexes was measured in sera of three patients, disclosing the values of 2.5-3.3 X 10(6) daltons for HBsAg complexes and 2.5-3.2 X 10(5) daltons for HBeAg complexes. Immune deposits consisting of hepatitis B virus antigen (HBeAg), IgG and C3 were detected in the glomeruli by immunoperoxidase and immunofluorescent assays respectively, in 3 out of 4 patients with membranous glomerulonephritis (MGN). No genetic defect of the complement system was found by the measurement of total haemolytic activity of complement and concentration of early complement components. From the analysis of clinical and laboratory data it was concluded that the appearance of HBeAg complexes correlated with more severe course of the disease.     

Isolation and partial characterization of circulating immune complexes in sera of children with HBV-mediated glomerulonephritis.

Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.     

Anti-SSB/La is one of the antineutrophil autoantibodies responsible for neutropenia and functional impairment of polymorphonuclear neutrophils in patients with systemic lupus erythematosus

Decreased number and impaired functions of polymorphonuclear neutrophils (PMN) due to the presence of anti-PMN autoantibodies in the serum render patients with systemic lupus erythematosus (SLE) susceptible to bacterial infections. However, the cognate antigens and pathological mechanisms of anti-PMN autoantibodies in SLE are rarely reported in the literature. In this study, we found approximately 20% of SLE sera contained anti-PMN autoantibodies detected by human PMN-coated cellular ELISA. A membrane protein with molecular weight of 50 kDa was identified as the cognate antigen of anti-PMN in Western blot after membrane-biotinylation and streptavidin column elution. The 50 kDa molecule was proved to be SSB/La after immunoscreening, molecular cloning and nucleotide sequencing of the gene from the human leucocyte cDNA library. Human anti-SSB/La autoantibodies purified from active SLE sera passing through the recombinant SSB/La conjugated Sepharose 4B affinity column could bind and penetrate into normal human PMN. Functional analysis revealed that the anti-SSB/La autoantibodies exerted a number of potent effects on human PMN, including suppressed phagocytosis, accelerated apoptosis and enhanced IL-8 production. These in vitro results suggest that anti-SSB/La is one of the anti-PMN autoantibodies capable of penetrating into PMN and responsible for neutropenia and functional impairment of PMN in patients with SLE.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM anti-idiotypes directed against anti-HBs molecules

A simple and specific enzyme-linked immunosorbent assay (ELISA) has been developed to detect circulating IgG and IgM anti-idiotypic antibodies directed against anti-HBs molecules using 96-well polyvinyl microtitre plates as the solid phase and HRPO-labelled goat anti-HBs as conjugate. Anti-idiotype reactions were observed in the supernatant portion after precipitation of immune complexes from sera with polyethylene glycol 6000 (PEG). Both IgG and IgM with anti-idiotype activity were detected concurrently in HBsAg-positive sera from HBV-infected patients and asymptomatic HBV carriers. Anti-idiotype activity was absent in HBsAg-negative sera from healthy persons, and in patients with non-A, non-B hepatitis and viral hepatitis A. However, such antibodies could be demonstrated in the sera of two out of eight HBsAg vaccine recipients negative for anti-HBs but in none of 11 recipients positive for anti-HBs after receiving a booster immunising dose of HBsAg vaccine. Those sera showing positive anti-idiotype reactions were free from rheumatoid factor and HBsAg/IgM or HBsAg/IgG complex activity. An analysis of anti-idiotype positive sera for anti-HBs, HBeAg and HBV-specific DNA-polymerase activity demonstrated these markers in 20%, 30% and 60% of cases, respectively. The presence of anti-idiotypic antibodies was presumed to permit a more active multiplication of hepatitis B virus.     

单克隆的抗-HBs在同系鼠体内诱导抗-HBs的观察

作者采用BALB/c鼠的抗-HBs单克隆抗体(Ab_1)作为抗原,免疫同系BALB/c小鼠,使其在自身体内产生Ab_2,观察了由Ab_2诱导的Ab_3的消长情况。经测定其血清,免疫小鼠在第7天出现Ab_2,随后第9天、第19天出现Ab_2的显著增高。Ab_3的出现是在第9天,随后在第11天,第17天和第21天出现增高现象。Ab_2与Ab_3的量的变化,呈周期性消长。一般说来,当Ab_2增高时,Ab_3减低;若Ab_2降低,则Ab_3增高。实验过程中,还发现Ab_3能与外界进入体内的HBsAg结合。上述实验中出现的Ab_2和Ab_3之间的相互刺激和相互中和的现象,表明是小鼠体内自身的抗个体型抗体的调节作用的结果,因而出现了上述周期性的动态变化,支持体内存在着一个个体型——抗个体型免疫网络的理论,本文依据实验事实,认为传统的被动免疫方法,在一定条件下,也能诱导出主动免疫。     

HBsAg基因疫苗诱导小鼠抗体产生的观察

将携带编码乙型肝炎病毒(HBV)表面抗原主要蛋白的S基因的质粒,直接注射小鼠肌肉中,小鼠4周后产生抗-HBs,阳转率83.3%(5/6),加强组阳转率66.7%(2/3),未加强组阳转率为100%(3/3);两组小鼠抗体持续时间可达28周以上;4周以后小鼠体内未检测到HBsAg.     

A Human Monoclonal IgG1λ Anti-Hepatitis B Surface Antibody

Peripheral blood mononuclear cells from a donor with a high titre of anti-hepatitis B surface (HBs) antibodies were fused with a cell line that was positive for Epstein-Barr virus nuclear antigen and sensitive to hypoxanthine-aminopterine-thymidine. A cell line was established that produces a monoclonal IgG1 lambda anti-HBs antibody. Afterwards, it appeared that the anti-HBs antibody-producing cell line had arisen from Epstein-Barr virus transformation of the donor B cells. The cell line is capable of producing up to 60 micrograms/ml of the monoclonal antibody, which has a high avidity for HBs antigen (Ag) and recognizes both ad and ay subtypes. The antibody is useful as a reagent for the detection of HBsAg in human serum. Over 1000 patient sera have been tested with a conventional third-generation assay in parallel, and only a single discrepant serum was found.     

IgG subclass composition of antibodies to HBsAg in circulating immune complexes from patients with hepatitis B virus infections

The IgG subclass of antibody associated with hepatitis B surface antigen (HBsAg) in circulating immune complexes (CIC) from patients with either acute or chronic hepatitis B virus (HBV) infections was measured using an isotype and antigen-specific ELISA. All patients were HBsAg positive but were negative for free anti-HBs antibody. The subclass of antibody associated with HBsAg in CIC in both groups was predominantly IgG1 and IgG4. This is in contrast to free anti-HBs in convalescent sera from patients recovering from HBV infection, which are highly restricted to IgG1 and IgG3. The finding of high levels of IgG4 antibodies in CIC suggest that CIC containing this subclass may be cleared less efficiently than CIC containing antibodies of other subclasses. Formation of these CIC may be an important factor in the progression of infection to chronicity and may also be involved in the antigen-specific immunosuppression seen in early acute and chronic HBV infections.