Auto-anti-anti-DNA antibodies from SLE patients and normals

Cross reacting auto-anti-idiotypic antibodies against anti-ssDNA antibodies were investigated in patients with systemic lupus erythematosus (SLE) and normals. Sera or immunoglobulins from SLE and normals depleted of anti-ssDNA activity and DNA antigen inhibited the reaction between 125I-F(ab')2 anti-ssDNA and ssDNA. In addition, binding to F(ab')2 portions of chromatographically purified portions of anti-ssDNA coated on polystyrene wells could be measured both in depleted SLE and normal sera. Depleted sera from SLE had both greater inhibitory activity and more antibody binding capacity than depleted sera from normals. IgG from SLE sera bound to both F(ab')2 anti-ssDNA and SLE F(ab')2 non-anti-ssDNA. However, the binding of IgG to F(ab')2 anti-ssDNA was significantly inhibited by ssDNA. These results indicate that cross reacting auto-anti-anti-ssDNA as well as other antibodies to F(ab')2 portions of homologous IgG are found in higher concentration in SLE than in normals.     

Identification of autoantibodies specific for the neutrophil adhesion glycoproteins CD11b/CD18 in patients with autoimmune neutropenia.

Anti-neutrophil antibodies have been described in a variety of clinical conditions associated with neutropenia. However, relatively little is known about the antigenic specificities of naturally occurring anti-neutrophil autoantibodies. We investigated the possibility that anti-neutrophil antibodies specific for the neutrophil adhesion glycoprotein (GP) complex CD11b/CD18 might be present in the sera of some patients with autoimmune neutropenia. These membrane GPs have been shown to be highly immunogenic in the production of murine monoclonal antibodies against neutrophil antigens. Moreover, autoantibodies to the platelet membrane GP complex IIb/IIIa, another member of the integrin family of cell adhesion proteins, have been demonstrated in immune thrombocytopenic purpura. Sera from 50 patients known to have anti-neutrophil IgG antibodies were evaluated using an immunobead "antigen capture" assay, modeled after a method used to identify anti-platelet GPIIb/IIIa autoantibodies. This assay detected anti-CD11b/CD18 autoantibodies in seven of the 50 sera. Each of these seven sera demonstrated decreased IgG binding to the neutrophils of a patient with congenital deficiency of CD11b/CD18. The patient with the highest levels of anti-CD11b/CD18 suffered recurrent skin infections and cellulitis, and died of respiratory failure during one of multiple episodes of pneumonia. Purified IgGs from five of these patients demonstrated effects on adhesion and/or opsonin receptor-mediated functions when tested with intact neutrophils in vitro. Our findings indicate that some patients with autoimmune neutropenia have autoantibodies specific for the functionally important neutrophil adhesion proteins CD11b/CD18. Our findings also raise the possibility that these autoantibodies may, in some cases, interfere with neutrophil function, thereby amplifying the risk of infection associated with neutropenia.     

Antibodies to myeloid precursor cells in autoimmune neutropenia

Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony- forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.     

Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects.

We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.     

Analysis of antibody to neutrophils associated with autoimmune neutropenia: possible recognition of Fc-receptor-related molecules

The serum of a 50-year-old male with neutropenia and a bout of bacterial infection was studied. Anti-neutrophil IgG antibody was detected by indirect immunofluorescence using a laser flow cytometry system. Purified IgG from our patient's serum did not inhibit chemotaxis of neutrophils, but inhibited rosette formation of neutrophils with ox red blood cells (ORBC) coated with anti-ORBC rabbit IgG dose-dependently. Surface iodination of neutrophils followed by their immunoprecipitation by purified IgG and sodium dodecylsulfate polyacrylamide gel electrophoresis showed a single band that corresponded to 45 kilodaltons. Possibly the IgG antibody in our patient's serum recognizes molecules related to Fc receptors.     

Serum platelet-reactive IgG of autoimmune thrombocytopenic purpura patients is not F(ab')2 mediated and a function of storage.

Serum platelet-reactive and glycoprotein (GP) IIb-GPIIIa-reactive IgG and F(ab')2 was examined in 39 patients with classic autoimmune thrombocytopenic purpura (ATP), two patients with anti-PLA1 antibody and 25 control subjects in an enzyme-linked immunosorbent assay. IgG was purified by diethyl aminoethyl chromatography and centrifuged at 100,000g before testing of the supernatant. Significant IgG binding (threefold to fourfold control IgG binding) was noted with 8 of 17 ATP patients' IgG, 2 anti-PLA1 IgGs, and 2 ATP patients with multiple platelet transfusions. However, F(ab')2 fragments of nine of nine positive ATP IgGs were nonreactive; F(ab')2 from the two anti-PLA1 and two multiply transfused ATP IgGs were as reactive as their intact IgG. Antiplatelet or anti-GPIIb-GPIIIa reactivity of ATP IgG could be adsorbed to fixed platelets or solid-phase GPIIb-GPIIIa and eluted with 0.1 mol/L glycine, pH 2.5. However, binding of IgG to GPIIb-GPIIIa could not be inhibited with F(ab')2 of ATP IgG or Fc fragments of control subjects. When platelet- or GPIIb-GPIIIa-reactive ATP IgG was applied to a Sephacryl 300 gel filtration column, no reactivity was noted in the 7S region, whereas anti-PLA1 localized to this region. Antiplatelet or anti-GPIIb-GPIIIa reactivity was noted in the void volume and accompanied by a high molecular weight protein region. An immunoblot of the void volume fraction with goat antihuman IgG (gamma chain) antibody showed high molecular weight bands greater than 250 Kd, which after reduction converted to a 55-Kd heavy-chain band. Fresh samples of ATP and control IgG processed within 1 to 2 days of blood withdrawal had no reactivity for GPIIb-GPIIIa. After storage at -20 degrees C for greater than 3 months, 5 of 19 ATP IgG became reactive, whereas 16 of 16 controls were nonreactive. Thus, platelet-reactive IgG of ATP sera appears to be caused by the development of IgG aggregates held together by disulfide bonds that develop on storage, and is not F(ab')2 mediated.     

The specificity of antibodies to the F(ab')2 fragment of human IgG

The specificity of IgG anti-F(ab')2 antibodies was examined by unfractionated sera of patients with rheumatoid arthritis and also with affinity-purified antibody preparations. Examination of the sera by an enzyme-linked immunosorbent assay, using pooled human F(ab')2 fragments absorbed to microtiter plates, revealed that IgG anti-F(ab')2 antibodies cross-react with human and rabbit IgG and rabbit F(ab')2. IgG anti-F(ab')2 antibodies were purified by affinity chromatography and, when tested by a fluid-phase inhibition enzyme-linked immunosorbent assay, were found to be of 2 types. One fraction, similar to pepsin agglutinator, reacted with human F(ab')2 fragment alone. The other fraction was cross-reactive with human IgG and yet failed to react with idiotopes on Fab or epitopes on Fc fragments. The IgG anti-F(ab')2 antibodies we purified had no reactivity toward a human immune complex prepared from tetanus toxoid and antitoxoid.     

Anti-neutrophil antibodies in inflammatory bowel disease: prevalence and diagnostic role.

Anti-neutrophil antibodies have been shown in sera from patients with a variety of inflammatory diseases. Those reacting with components of neutrophil cytoplasm are associated with systemic vasculitis. Both nuclear and perinuclear staining patterns on human neutrophils have been reported using sera from patients with inflammatory bowel disease. We have evaluated the reactivity against human neutrophils of sera from 100 patients with inflammatory bowel disease, 14 disease controls, and 20 normal volunteers. Altogether 27/50 (54%) sera from patients with ulcerative colitis contained antibodies that reacted with cytospun ethanol fixed neutrophils compared with 5/50 (10%) from Crohn's disease (p less than 0.001) and 0/34 control sera (p less than 0.001). All seven sera from patients with proctitis alone were negative (p less than 0.01). There was no correlation between presence or titre of anti-neutrophil antibodies and either disease activity or treatment. Positive sera gave three different staining patterns on human neutrophils. The predominant pattern was perinuclear (17/32); 12 sera gave a cytoplasmic and three a homogeneous nuclear staining pattern. None of the patients or the controls had antibodies to myeloperoxidase, elastase, or serine proteinase 3, all of which are recognised by anti-neutrophil cytoplasmic antibodies. Only 2/27 sera positive by indirect immunofluorescence reacted with an extract of neutrophil primary granules. In conclusion, anti-neutrophil antibodies occur more commonly in ulcerative colitis than in Crohn's disease or control subjects and the anti-neutrophil antibodies found in inflammatory bowel disease are different from those associated with vasculitis.     

Chronic neutropenia of childhood: frequent association with parvovirus infection and correlations with bone marrow culture studies

Summary. Children with neutropenia of more than 3 months duration often have evidence of immune-mediated destruction of mature neutrophils and variable abnormalities of myeloid precursors in their bone marrow. These patients often have anti-neutrophil antibodies which persist for several months. To further investigate the aetiology of neutropenia in such patients, bone marrow cells were evaluated for the presence of common viruses. Fifteen of 19 patients tested had evidence for parvovirus infection by PCR amplification of bone marrow DNA with parvovirus specific primers. Of these 15, six also had serologic evidence of parvovirus infection. Anti-neutrophil antibodies were identified in nine of 12 patients with parvovirus infection. Bone marrow culture studies done on six patients revealed varying degrees of myeloid and erythroid inhibition by patient plasma. These studies indicate that parvovirus may be a common cause of immune-mediated neutropenia in children.     

Characterization of multiple quinine-dependent antibodies in a patient with episodic hemolytic uremic syndrome and immune agranulocytosis.

A 23-year-old woman experienced six distinct episodes of severe combined neutropenia and thrombocytopenia. At least one of the episodes was accompanied by hemodialysis-requiring acute renal failure and fragmentation hemolysis (hemolytic uremic syndrome [HUS]). In retrospect, all episodes were probably associated with the ingestion of quinine. Quinine-dependent antibodies to platelets, neutrophils, T lymphocytes, and red blood cells (RBCs) were detected in the patient's serum. By a monoclonal antibody antigen capture assay, the patient's serum contained IgG antibodies, which in the presence, but not absence, of quinine reacted with platelet glycoprotein (GP) complexes Ib/IX and IIb/IIIa, but not Ia/IIa. By immunoprecipitation assay, the serum, after addition of quinine, reacted strongly with an 85-Kd neutrophil membrane protein and weakly with 130- and 60-Kd moieties. Serum adsorbed with RBCs in the presence of quinine continued to react with platelets and neutrophils, and serum that was absorbed with platelets continued to react with neutrophils and RBCs, indicating that the antigenic targets were different on platelets, neutrophils, and RBCs. Since platelets and endothelial cells share some antigens, we tested patient serum for antibodies to human umbilical vein endothelial cells (HUVEC); no quinine-dependent antibodies to HUVEC were detected. However, her quinine-dependent antibodies not only bound to platelets and neutrophils, but also activated neutrophils. Thus, the patient's serum with quinine aggregated neutrophils, but neither agent alone caused activation. Moreover, the patient's serum with quinine (but not without) augmented the adherence of neutrophils to HUVEC. Treatment of the patient's serum with staphylococcal protein A removed the quinine neutrophil aggregation cofactor, suggesting it was due to IgG. In both neutrophil aggregation and adherence assays, decomplementation of the patient's serum markedly blunted its effect. Furthermore, the patient's serum failed to aggregate formalin-inactivated neutrophils, suggesting neutrophil activation, probably by activated complement, was necessary for aggregation and adhesivity to endothelium. We conclude that our patient's neutropenia, thrombocytopenia, lymphopenia, and anemia were due to quinine-dependent antibodies, and that these antibodies recognized epitopes that were different in the three target cell populations. We further suggest that HUS was likely secondary to the activation and adhesion of neutrophils to endothelium.     

Drug-dependent and non-drug-dependent antiplatelet antibody in drug-induced immunologic thrombocytopenic purpura

The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid-phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug-dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.     

Felty syndrome: autoimmune neutropenia or immune-complex-mediated disease?

Summary Immunofluorescence on polymorphonuclear cells (PMN) of patients with Felty syndrome (FS) revealed increased amounts of IgG, IgA, and IgM bound to the PMN surface compared with PMN of patients with rheumatoid arthritis alone. A positve correlation was found between the score for surface-bound immunoglobulins on FS-PMN and the results of the C1q binding assay in FS sera. After preincubation with sera from 20 patients with FS, immunofluorescence on PMN from healthy controls (HC) showed that these cells had bound IgG, IgA, and IgM. However F(ab')₂ fragments of IgG from FS sera did not bind to PMN, although the antigen-binding reactivity of the F(ab')₂ fragments was maintained as shown by control experiments. Immunoglobulins eluted from FS-PMN failed to bind to HC-PMN, whereas the corresponding IgG of patients with autoimmune neutropenia was bound. Gel filtration of FS sera on Sepharose 4B showed that the binding of IgG in FS sera to PMN did not coincide with the 7S peak but occurred mainly in fractions containing larger material. No binding of IgA and IgM to HC-PMN was found after incubation with FS sera pretreated with polyethylene glycol (PEG) to precipitate immune complexes. These results indicate that in sera of patients with FS the PMN-binding reactivity of IgG, IgA, and IgM is due to the binding of immune complexes containing these immunoglobulins and not to presence of autoantibodies directed to antigens on the neutrophil surface.     

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.     

Is alpha-actinin a target for pathogenic anti-DNA antibodies in lupus nephritis?

OBJECTIVE: Following recent reports that pathogenic murine anti-DNA antibodies bind to alpha-actinin, it was obviously of interest to assess the ability of human pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies to bind this antigen. Both human monoclonal anti-DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated. METHODS: An enzyme-linked immunosorbent assay was established to measure immunoglobulin binding to alpha-actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high-affinity human anti-dsDNA IgG hybridomas (RH14, B3, and DIL-6) and 7 human IgM anti-DNA hybridomas was also investigated. RESULTS: A greater proportion of anti-dsDNA IgG-binding antibodies purified from patients with renal disease bound to alpha-actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100-kd alpha-actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to alpha-actinin, while nonpathogenic DIL-6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to alpha-actinin. CONCLUSION: The results of our study support the findings of previous studies using murine anti-DNA monoclonal antibodies, which suggest that pathogenic anti-dsDNA antibodies cross-react with alpha-actinin.     

In vitro killing of neuroblastoma cells by neutrophils derived from granulocyte colony-stimulating factor-treated cancer patients using an anti-disialoganglioside/anti-Fc gamma RI bispecific antibody

Neutrophils isolated from cancer patients treated with granulocyte colony-stimulating factor (G-CSF) express high levels of Fc gamma RI. They exhibited an efficient killing of GD2+ neuroblastoma cells in the presence of an antidisialoganglioside (GD2) mouse monoclonal antibody (MoAb; 7A4, IgG3 kappa). However, this cytotoxicity was totally blocked by human monomeric IgG. In contrast, a bispecific antibody (7A4 bis 22/MDX-260), prepared by chemically linking an F(ab') fragment of 7A4 with an F(ab') fragment of an anti-Fc gamma RI MoAb, 22, which binds outside the Fc binding domain, triggered antibody-dependent cell cytotoxicity, even when neutrophils were preincubated with human monomeric IgG. F(ab')2 22 MoAb abrogated the MDX-260 killing without affecting that of 7A4. The 3G8 MoAb, directed against the Fc gamma RIII binding site, did not inhibit the cytotoxicity induced by either antibody. Thus, these results indicate that G-CSF-activated neutrophils exert their cytotoxic effect against neuroblastoma cells through Fc gamma RI and not Fc gamma RIII, and that the saturation of the high affinity Fc gamma RI by monomeric IgG can be overcome by the use of bispecific antibodies binding epitopes outside the IgG Fc gamma RI binding site. A combined administration of such bispecific antibodies and G-CSF may be, therefore, an efficient therapeutic approach to trigger tumor lysis by cytotoxic neutrophils in vivo.     

Anti-La/SSB antibody is present in some normal sera and is coincident with anti-Ro/SSA precipitins in systemic lupus erythematosus

Sera from 42 patients with systemic lupus erythematosus (SLE) and precipitating antibody to the La/SSB antigen contained 80,000 to 28,800,000 times the minimum detectable binding activity (units) in a solid phase ELISA assay using purified La/SSB antigen. Three normal sera (of 40 tested) had 7,200,21,600 and 21,100 units, respectively, while the log average of the 40 normals was 270 units. Binding activity in normal sera was in the F(ab')2 fragments of IgG, inhibited by purified La/SSB antigen, and bound the major La/SSB peptides. The average apparent relative binding affinity to La/SSB in normal donors was 10-fold lower than in SLE patients with anti-La/SSB precipitins. Analysis of other SLE sera revealed a high incidence of anti-La/SSB in association with anti-Ro/SSA precipitins.     

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.     

Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of glycoproteins Ib, IIb, IIIa, and IX

The molecular nature of platelet receptors for quinine- and quinidine- dependent antiplatelet antibodies (Q.Ab and Qd.Ab) was studied by immunoblotting. One Q.Ab caused quinine-dependent IgG binding to platelet proteins with molecular weights (mol wts) of 174 Kd and 93 Kd and another to only a 93-Kd protein. A third Q.Ab caused binding to 174- , 140-, 93-, and 57-Kd proteins, while a fourth Q.Ab and a Qd.Ab caused IgG binding to 174- and 18-Kd proteins. Using platelets from patients with Glanzmann's thrombasthenia or Bernard Soulier syndrome and purified GPIIIa, these proteins were shown to be GPIb, GPIIb, GPIIIa, GPIX, and an unidentified 57-Kd protein missing in Bernard Soulier syndrome. Binding to the 93-Kd protein was independent of the PIA1 antigen. Absorption of one Q.Ab with Glanzmann's thrombasthenia platelets revealed different populations of antibodies with different specificities within the one patient. Thus Q.Ab and Qd.Ab are heterogeneous and may be directed toward different epitopes on major platelet glycoproteins.     

Antibodies to granulocyte precursors in selective myeloid hypoplasia and other suspected autoimmune neutropenias: use of HL-60 cells as targets

Patients with syndromes of autoantibody-mediated hematocytopenias may manifest signs of increased cell destruction and/or decreased cell production, depending on the maturity of the target cell and the effects of antibody binding. The purpose of this study was to use a cultured human cell line of hematopoietic origin for in vitro assays of antibody binding to overcome the relative inaccessibility of natural human marrow progenitor cells. This report describes the detection, using radioiodinated staphylococcal protein A (SPA), of antibodies binding to a human promyelocytic cell line (HL-60) in sera from three patients with chronic idiopathic granulocytic hypoplasia ("pure white cell aplasia," PWCA) and 22 patients with other syndromes of suspected immune neutropenia. Bone marrow from patients with increased IgG binding to HL-60 cells showed less than 15% granulocytic lineage cellularity in 11 of 17 cases. In vitro differentiation of HL-60 cells by retinoic acid resulted in increased IgG binding for sera that had shown increased IgG binding to mature granulocytes but not undifferentiated HL-60 cells; in contrast, for sera with antibodies to untreated HL-60 cells and for normal serum, in vitro differentiation had little effect on IgG binding. Antibodies eluted from mature granulocytes were similar to the parent serum regarding the ratio of IgG binding to mature cells v HL-60 cells. No sera from 19 patients with febrile transfusion reactions showed increased IgG binding to HL- 60 cells in the absence of increased IgG binding to mature granulocytes, although two sera had antibodies to both cell types. The use of HL-60 cells as targets may permit measurement of serum antibodies associated with granulocytic hypoplasia. In combination with assays to detect antibody binding to mature granulocytes, these techniques may discriminate among autoantibody specificities for antigens that are gained, conserved, or lost during myeloid maturation.     

Western blot identification of platelet proteins that bind normal serum immunoglobulins. Characteristics of a 95-Kd reactive protein

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2- mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.