Anti-granulocyte opsonic activity and autoimmune neutropenia

Sera from patients with unexplained neutropenia have been assayed for anti-granulocyte opsonic activity using a chemiluminescence technique which measures the metabolic response of human monocytes to antibody-coated granulocytes. This rapid and simple technique was more sensitive than indirect immunofluorescence in the detection of anti-granulocyte antibodies. Anti-granulocyte opsonic activity was detected in sera from 17 of 31 patients, suggesting that their neutropenia may have had an autoimmune basis. The opsonic activity of five of the 17 sera was increased when granulocytes were sensitized in the presence of fresh serum. Four of these sera bound IgM and C3b to granulocytes in the immunofluorescence test. Human IgG when added to the monocyte suspension medium inhibited monocyte response to IgG antibody-opsonized granulocytes. This inhibition was less when granulocytes were opsonized with sera containing IgM and complement granulocyte-binding activity. This observation may be relevant to the selection of neutropenic patients for therapeutic use of intravenous immunoglobulin.     

Impaired tuftsin activity in cirrhosis: relationship with splenic function and clinical outcome

BACKGROUND: Cirrhotic patients show increased susceptibility to bacterial infections. It is not known whether tuftsin deficiency, which is associated with an increased incidence of infections in many disease states, is present in cirrhosis. Our aims were to determine whether tuftsin activity is deficient in cirrhosis and if so, whether this deficiency is related to splenic function, contributes to altered neutrophil granulocyte function, or influences the occurrence of bacterial infections and patient survival. METHODS: Tuftsin activity and splenic function were assessed in 31 patients with liver cirrhosis and 31 healthy subjects. The phagocytic activity of neutrophil granulocytes from 23 patients was tested in vitro with addition of both autologous and pooled sera from healthy subjects. In 10 patients and eight controls it was also tested with addition of synthetic tuftsin. Patients were followed up until death or liver transplantation. RESULTS: Patients had reduced tuftsin activity (median 8% (range 3-24.5)) compared with controls (17% (11.5-37)) (p<0.001) and a higher pitted red cell count (p<0.001). Tuftsin activity was correlated with pitted cell count (p=0.02) and the Child-Pugh score (p=0.002). Nineteen of 23 patients showed deficient phagocytic activity of neutrophil granulocytes, which was correlated with tuftsin activity (p<0.001), improved in all cases but one with addition of serum from healthy subjects, and normalised with addition of synthetic tuftsin. Reduced tuftsin activity did not influence patient survival but was associated with a higher incidence of bacterial infections (p=0.029). COMMENT: Tuftsin activity was reduced in cirrhosis, and contributed to impaired phagocytic activity of neutrophil granulocytes. Such an abnormality appears to be related to impaired splenic function and severity of cirrhosis, and probably favours the occurrence of bacterial infections.     

Chronic idiopathic and secondary neutropenia: clinical and serological investigations

Clinical data on 49 patients with chronic idiopathic neutropenia (CIN) and 42 patients with neutropenia secondary to a well-defined immunological disorder (SN) were collected and related to serological parameters. In 47% of the patients with CIN and 53% of those with SN, a positive direct immunofluorescence test was obtained with granulocytes from the patients. In the sera from the patients in the two groups, antibodies against donor granulocytes were detected by the indirect immunofluorescence test, the leucoagglutination test and/or the granulocytotoxicity test in 15%, 19% and 15%, respectively. The results of the above tests could not be correlated with any clinical or haematological parameter. Immune complexes in the serum were detected by the 125I-Clq-binding test in 29% of patients with CIN and in 58% of those with SN. The presence of serum immune complexes correlated well with the existence of a low neutrophil count, but not with the presence of recurrent infections, with bone-marrow abnormalities, or with positive reactions in other serological tests. The sera of eight out of 14 patients with CIN and seven out of 12 patients with SN had inhibitory activity for myeloid colony formation in vitro (CFU-GM). This CFU-GM inhibitory activity was correlated with the presence of recurrent infections and with hypoplasia of the myeloid compartment of the bone marrow, but not with positive reactions in other tests. We conclude that the 125I-Clq-binding test probably detects circulating immune complexes that induce a shift neutropenia, whereas serum activity inhibitory for CFU-GM possibly relates to clinically more serious forms of neutropenia. The significance of neutrophil-bound Ig and granulocyte-reactive antibodies in the serum is not clear.     

Contribution of immunofluorescence to the identification and characterization of anti-neutrophil cytoplasmic autoantibodies. The role of different fixatives

OBJECTIVE: To study the sera from selected groups of antineutrophil cytoplasmic antibody (ANCA) positive patients by means of the indirect immunofluorescence test (ANCA-IIF) with different fixatives, in order to better discriminate among the various ANCAs (Ag-specificity and disease associations), especially those for which the antigen targets have not yet been identified. METHODS: Eighty pathological serum samples and 15 normal sera were evaluated. Pathological samples included sera from 30 ulcerative colitis (UC) ANCA positive patients, 30 P-ANCA/myeloperoxidase (MPO-ANCA) positive microscopic polyangiitis (MPA) patients, 10 C-ANCA/proteinase 3 (PR3-ANCA) positive Wegener's granulomatosis (WG) patients, and 10 antinuclear antibody (ANA) positive (ANCA negative) systemic lupus erythematosus (SLE) patients. ANCA were detected by IIF on ethanol, methanol and formalin-fixed granulocytes and by ELISAs specific for MPO, PR3, lactoferrin (LF) and bactericidal/permeability-increasing protein (BPI). Additionally, sera were tested for the presence of antinuclear antibodies on IIF. RESULTS: 96% of serum samples from UC patients, positive by IIF on ethanol-fixed granulocytes, became negative when tested on formalin-fixed neutrophil slides. On the contrary, 95% of sera from vasculitic patients showed a clear diffuse granular cytoplasmic pattern on the same substrate; sera from all 10 SLE patients did not show any reactivity when formalin was used as fixative. On methanol-fixed neutrophils, 100% of UC P-ANCA positive sera were positive with the same pattern versus only 20% of vasculitic P-ANCA positive (MPO positive). Methanol fixation had no effect on PR3-ANCA and ANA positive sera. CONCLUSION: The comparison of IIF patterns of sera tested on different fixed cells may be useful to distinguish vasculitis-related P-ANCA versus ANA and vasculitis-related P-ANCA versus UC-related P-ANCA.     

Amodiaquine-induced immune agranulocytosis

This report describes two patients who developed agranulocytosis while receiving prophylactic amodiaquine treatment. The neutrophil counts returned to normal in one after stopping the drug while the other died of sepsis. Amodiaquine-dependent circulating neutrophil IgG antibodies were demonstrated in both patients using the indirect granulocyte immunofluorescence test. The antineutrophil antibody activity was enhanced with the use of the major amodiaquine metabolite, mono-desethyl amodiaquine. Additional studies showed the activity of the sera to be nondialysable, heat stable, active against autologous as well as allogenic cells, and absent from the convalescent sera. There was no growth inhibition of allogenic myeloid committed progenitor cells (CFU-GM) following incubation with the patients' sera, complement and amodiaquine. These results indicate that agranulocytosis can be mediated by a drug-dependent antibody which affects mature blood cells.     

Autoantibodies to neutrophil cytoplasmic (ANCA) and endothelial cell surface antigens (AECA) in chronic inflammatory bowel disease

Sera from 103 patients with chronic inflammatory bowel disease (IBD) were tested prospectively for antibodies against neutrophil cytoplasmic antigens (anti-neutrophil cytoplasm antibodies, ANCA) and endothelial cell surface antigens (anti-endothelial cell antibodies, AECA) by indirect immunofluorescence (IIF) and assays based on whole fixed neutrophils, purified neutrophil enzyme substrates and human umbilical vein endothelial cells. Using IIF, ANCA were found in 26 IBD sera (25%) and in none of 51 controls. Twenty-two positive sera (85%) were from patients with ulcerative colitis (UC). The pattern of distribution of immunofluorescence was always perinuclear (P-ANCA). A majority of UC patients positive for these autoantibodies (68%) had active colitis, but none had evidence of vasculitis. Using a whole neutrophil ELISA, binding was demonstrable in 73% of UC sera compared to 27% of Crohn's (CD) sera and only 4% of controls. Unlike vasculitis sera, UC sera with P-ANCA did not bind strongly to myeloperoxidase (MPO). Forty-five per cent of IBD sera tested positive for IgG AECA in an endothelial cell ELISA, compared to seven of 51 (14%) controls. Binding correlated with both active and extensive colitis. A type of P-ANCA, in most cases distinct from MPO-specific P-ANCA observed in vasculitis, is detected in a significant proportion of patients with UC, but rarely Crohn's colitis and therefore may be of differential diagnostic value. IgG AECA are also frequent in CIBD sera but are less disease specific than ANCA. (Aust NZ J Med 1992; 22: 652ndash;659.)     

Anti-platelet opsonic activity in alloimmune and autoimmune thrombocytopenia

A chemiluminescence technique (CLT) has been developed which measures the interaction between human monocytes and antibody-coated (opsonized) platelets. This technique has an objective end-point, is simple to perform and is of comparable sensitivity to the platelet suspension immunofluorescence test (PSIFT) when used to detect anti-platelet allo-antibodies. In contrast, only 4/20 sera from patients with clinically diagnosed autoimmune thrombocytopenia were opsonic in the CLT, while 8/20 of these same sera bound IgG to platelets in the PSIFT. Only one serum gave positive results in both tests.     

系统性红斑狼疮患者循环抗皮肤基底膜带抗体及其与临床的相关性研究

目的了解抗皮肤基底膜带(BMZ)抗体在系统性红斑狼疮(SLE)患者循环中的存在情况,探讨其与临床的相关性。方法采用间接免疫荧光法检测70例SLE患者血清抗BMZ抗体,并分析其与SLE的临床表现、免疫指标的相关性。结果47例(67%) SLE患者存在循环抗BMZ抗体,其中IgG>IgM>IgA;抗体主要结合于盐裂皮肤的表皮侧,少数可结合于真皮侧或表、真皮两侧;伴皮肤损害的SLE患者循环抗BMZ抗体阳性率明显高于无皮肤损害者(P<0.05);病情处于活动期或伴肾脏损害、关节炎、脱发、光敏感、抗dsDNA抗体阳性的SLE患者与病情处于稳定期或不伴以上临床表现、抗dsDNA抗体阴性的患者比较,其抗BMZ抗体的阳性率差异无显著性。结论SLE患者循环中存在较高发生率的抗BMZ抗体,具有抗体/抗原的异质性;抗BMZ抗体的出现与SLE皮肤损害有关,而与病情活动、其他临床表现、抗dsDNA抗体无关;抗BMZ抗体可能参与了SLE皮肤损害的免疫机制。     

Sera from patients with systemic lupus erythematosus demonstrate enhanced IgG binding to endothelial cells pretreated with tumour necrosis factor alpha

Fluorescence flow cytometry and indirect immunofluorescence were used to detect circulating IgG antiendothelial cell antibodies (IgG-AECA) in the sera of patients suffering from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS). Pretreatment of endothelial cells with tumour necrosis factor alpha (TNF), but not with interferon gamma (IFN), increased the IgG binding from sera of some patients with active SLE. In contrast, no change in binding activity to cytokine-stimulated endothelial cells was observed in the RA and PSS sera. The results of this study suggested that the enhanced binding of IgG from the sera of patients with SLE to endothelial cells stimulated with TNF may be due to the ability of this cytokine to increase the expression of potential antigens on the surface of these cells. Hence, TNF may play a role in the immune-mediated vascular damage associated with SLE.     

Anti-neutrophil cytoplasmic antibody in patients with systemic lupus erythematosus is unrelated to clinical features

Summary To investigate the incidence, the specificity and clinical significance of positivity for serum anti-neutrophil cytoplasmic antibody (ANCA) in 31 patients with systemic lupus erythematosus (SLE), the indirect immunofluorescence (IIF) technique and enzyme-linked immunosorbent assay (ELISA) were used to measure ANCA. Purified myeloperoxidase (MPO), lactoferrin (LF), cathepsin-G (CTG) and elastase (HLE) served as ANCA antigens for ELISA. Thirteen (42%) of the 31 SLE patients showed positivity for perinuclear, but not cytoplasmic, ANCA by IIF. Five of 31 sera were positive for MPO, 10 for LF, 1 for CTG and 0 for HLE by ELISA. Patients positive for ANCA had a higher score of SLE disease activity index (SLEDAI) than those without ANCA. There was no correlation between ANCA positivity, clinical manifestations, or organic involvement. While the ANCA in patients with SLE reflected disease activity, it was unrelated to organic involvement.     

Granulocyte Function in Malignant Monoclonal Gammopathy

Granulocyte function was studied in 22 patients with untreated myelomatosis or macro-globulinaemia. Granulocyte adhesiveness (GA) and migration in capillary tubes (Tm) were, except for light chain disease, significantly decreased in patients of all gammopathy classes especially IgG myelomatosis. A plasma factor inhibited GA. The impairment of Tm was due to an inhibiting factor as well as lack of a stimulating plasma factor. Migration of granulocytes to skin chambers was decreased in patients with IgG myelomatosis. Chemiluminescence production during phagocytosis of opsonized zymosan was decreased. Compared to control sera, the opsonic activity of patient sera was strongly decreased. The impaired granulocyte functions in patients with malignant monoclonal gammopathy may contribute to the enhanced susceptibility to infections in these patients.     

Association of IgG Anti-Brain Antibodies with Central Nervous System Dysfunction in Systemic Lupus Erythematosus

Sera from 20 patients with systemic lupus erythematosus (SLE) and active central nervous system (CNS) dysfunction were examined by indirect immunofluorescence for antibodies to neuronal membrane determinants. Warm-reactive IgG antibodies were demonstrable in 82% (9/11) of patients with clinical evidence for seizures or diffuse CNS disease, but these antibodies generally were absent in non-CNS SLE sera or when focal neurologic deficit or psychosis was the primary CNS manifestation. Cold-reactive antibodies of the IgM class were equally prevalent in patients with or without CNS disease and appeared to be more directly correlated with extra-CNS systemic illness. Absorption experiments with lymphocytes, brain homogenate, and various other tissues suggested a predominant brainspecificity for IgG antibodies and partial lymphocyte cross-reactivity for IgM antibodies. Interpretations of this special association between IgG anti-brain antibodies and diffuse CNS dysfunction in SLE are discussed.     

Association of IgG anti-brain antibodies with central nervous system dysfunction in systemic lupus erythematosus.

Sera from 20 patients with systemic lupus erythematosus (SLE) and active central nervous system (CNS) dysfunction were examined by indirect immunofluorescence for antibodies to neuronal membrane determinants. Warm-reactive IgG antibodies were demonstrable in 82% (9/11) of patients with clinical evidence for seizures or diffuse CNS disease, but these antibodies generally were absent in non-CNS SLE sera or when focal neurologic deficit or psychosis was the primary CNS manifestation. Cold-reactive antibodies of the IgM class were equally prevalent in patients with or without CNS disease and appeared to be more directly correlated with extra-CNS systemic illness. Absorption experiments with lymphocytes, brain homogenate, and various other tissues suggested a predominant brain-specificity for IgG antibodies and partial lymphocyte cross-reactivity for IgM antibodies. Interpretations of this special association between IgG anti-brain antibodies and diffuse CNS dysfunction in SLE are discussed.     

Cytotoxic activity of cerebrospinal fluids (CSF's) against lymphocytes and phagocytes: comparison of normal and systemic lupus erythematosus CSF's.

Fifty cerebrospinal fluids (CSF), 24 normal, 26 from systemic lupus erythematosus (SLE) patients were tested for cytotoxic activity against human lymphocytes, granulocytes and monocytes. Normal and SLE CSF's frequently killed all 3 cell types. Lympho- and granulocytotoxins often reacted at both 4 degrees/24 degrees C, and at 37 degrees C. They were more active when no complement was added (p less than 0.01), whereas monocytotoxicity was complement-dependent (p less than 0.01). Normal CSF's more often contained cold-reacting lymphocytotoxins and SLE CSF's more often had warm-reacting monocytotoxins, but the differences were not significant (p = 0.03). Cytotoxins were easily absorbed to and eluted from lymphocytes and granulocytes, and when CSF's were toxic to both types of cells, the corresponding eluates usually retained this activity. Sometimes, only 1 type of cell was killed by the eluate, whereas cytotoxicity against another was retained by the corresponding supernatant. In SLE remarkable differences were noted between CSF cytotoxins and serum cytotoxins. The former were often more potent at 37 degrees C not requiring non-human complement. Preliminary characterization of CSF cytotoxins suggests they may be IgG, however, participation of non-Ig cytotoxic substances cannot be excluded.     

Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity

BACKGROUND: Increased numbers of apoptotic neutrophils, and impaired monocyte/macrophage clearance of apoptotic cells, have been demonstrated in systemic lupus erythematosus (SLE). CD44 is implicated in the clearance of apoptotic neutrophils. OBJECTIVE: To determine the expression of CD44 on peripheral blood monocytes and neutrophils in SLE, and examine the relations with disease activity and numbers of circulating apoptotic neutrophils. METHODS: Peripheral blood was sampled from 31 patients with SLE, 19 healthy normal subjects, and 19 patients with rheumatoid arthritis (RA). Monocyte and neutrophil density of surface CD44 expression was determined by immunofluorescence labelling and flow cytometry, and results expressed as mean channel fluorescence (MCF) values. Neutrophil apoptosis was measured by morphology in 15 patients with SLE, nine with RA, and six normal subjects. RESULTS: Monocyte CD44 expression was significantly lower in SLE (median MCF 4.71) than in healthy normal subjects (median MCF 5.61) and controls with RA (median MCF 5.39). Neutrophil CD44 expression was also significantly lower in SLE (median MCF 1.95) than in healthy normal subjects (median MCF 2.37) and controls with RA (median MCF 2.60). Monocyte, but not neutrophil, CD44 expression correlated negatively with the percentage of apoptotic neutrophils. There was no significant correlation of monocyte or neutrophil CD44 expression in SLE with disease activity or damage. CONCLUSIONS: Monocyte and neutrophil CD44 expression is reduced in SLE, and this may contribute to the impaired recognition and clearance of apoptotic neutrophils by monocyte derived macrophages.     

Opsonic activity of myeloma immuno-globulins (mm-lg)

Patients with MM are at an increased risk for life-threatening bacterial infections, primarily by organisms that require opsonization for interaction with granulocytes. In the present study we used a neutrophil chemiluminescence (CL) assay of opsonization to explore the opsonic activity of MM-lg for zymosan particles. Particles were treated with serum lacking in Ig to explore the contribution of Ig to zymosan opsonization. This serum was found to have 69 ± 2.6% (X? ± SEM) of the opsonic activity of normal serum (P < .001). Normal serum concentrations of normal IgG, MM-IgG, and MM-IgA all lacked opsonic activity for zymosan. However, when particles were treated with Nl-IgG, MM-IgG, or MM-IgA as well as complement, normal opsonic activity was generated. Increasing the concentration of the Ig to stimulate MM serum did not inhibit this opsonic activity. Thus both MM-IgG and MM-IgA can function as normal, nonspecific opsonins.     

Inhibition of Immune Phagocytosis by Human Sera with HLA A, B, C and DR but Not with DQ or EM Type Reactivity

613 sera from pregnant women were investigated for inhibition of immune phagocytosis (IPI) by monocytes exposed to anti-D (Rh)-sensitized human red blood cells. IPI was detected in 42 (26%) of 165 and in 78 (17%) of 448 sera assessed against monocytes from 15 or 5 panel donors, respectively. All IPI-positive sera reacted in an allotypic pattern. Eight IPI-positive sera tested with autologous monocytes were found to be nonreactive. Upon comparative analysis of 448 sera, a significant correlation was found between the specific patterns of IPI and lymphocytotoxicity by HLA antibodies. In addition, 13 sera (4%) were positive for IPI, but not for lymphocytotoxicity. Twenty IPI-positive sera were tested by indirect immunofluorescence against platelets and T lymphocytes and revealed IgG antibodies in 16 and 11 sera, respectively. IPI activity could be removed from 8 out of 10 positive sera by platelet pool absorption. While virtually all sera exhibiting HLA, A, B or C-like activity (cytotoxicity with all cells) or HLA DR-like activity (exclusive cytotoxicity with B lymphocytes and monocytes) were IPI-positive, IPI was infrequently observed with sera containing HLA DQ-like activity (cytotoxicity with B lymphocytes only). IPI was also rarely seen with sera cytotoxic only to monocytes and not at all with sera containing antibodies against endothelial/monocyte antigens. We conclude that IPI is caused by cytotoxic as well as noncytotoxic HLA A, B, C, DR-specific antibodies. This effect may bear significance for the maternal immune response against fetal antigens and may be useful for pretransplant histocompatibility testing.     

Development of surface antigen during maturation of bone marrow neutrophil granulocytes.

The presence of neutrophil surface antigens on maturing bone marrow granulocytes was examined by indirect immunofluorescence and cellular immunoadsorption using heterologous antibody to mature neutrophil granulocytes. The results show that bone marrow neutrophils possess surface antigens that appear during cell maturation from myeloblasts. Fluorescent antibody capping and patching, indicators of antigen mobility, were more pronounced in mature than in immature cells. Neutrophil surface antigen development could also be demonstrated during granulocyte maturation in vitro.     

Serum opsonins to serogroup B meningococci in meningococcal disease

The opsonic activity to serogroup B meningococci (B:15:P1.16) was measured in sera from 101 patients with meningococcal disease using a chemiluminescence method. On admission to hospital the opsonic activity was lower in 12 patients who died than in survivors (p = 0.0007). A close association was observed between the opsonic activity and the duration of symptoms before admission, the severity of the disease, and the levels of IgG antibodies to the outer membrane complex (15:P1.16). The opsonic activity was low in 2 premorbid sera compared to healthy controls. The mean opsonic activity peaked 2 weeks after admission and was still high 3-5 years later. Meningococcal strains of different serogroups, serotypes and subtypes induced a similar increase in opsonic activity to B:15:P1.16 meningococci. No increase in activity was observed in sera from patients with meningitis and septicemia caused by other bacteria. Serum opsonins seem to be of significant importance in the host defence against serogroup B meningococci.