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.     

Lack of evidence for the presence of neutrophil autoantibodies in the serum of patients with Felty''s syndrome

Sera of 22 patients with Felty's syndrome and 14 patients with rheumatoid arthritis were tested in assays routinely used for the detection of neutrophil antibodies (i.e. immunofluorescence, agglutination and cytotoxicity tests) as well as in the antibody-dependent lymphocyte-mediated granulocytotoxicity test. One or more of the routinely used assays were positive in a high percentage of the sera (77% and 64%, respectively). The antibody-dependent lymphocyte-mediated granulocytotoxicity test was only positive with sera of three patients with Felty's syndrome. Positive results in the immunofluorescence and agglutination tests could be attributed to the presence of immune complexes in the sera, whereas positive antibody-dependent lymphocyte-mediated granulocytotoxicity tests were probably due to the presence of HLA alloantibodies. It is concluded that serological tests routinely used for the detection of neutrophil autoantibodies should be interpreted with caution in patients with Felty's syndrome. Our results also indicate that the neutropenia in Felty's syndrome is rarely, if ever, due to neutrophil-specific autoantibodies.     

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.     

Serological studies in patients on platelet- and granulocyte-substitution therapy

S ummary . A serological follow-up study was undertaken in 47 patients with bone-marrow failure, who were repeatedly transfused with random donor granulocytes and/or platelets. Sera, obtained at regular intervals, were investigated in the leucoagglutination test, the lymphocytotoxic test and the immunofluorescence test on paraformaldehyde-fixed platelets, granulocytes and lymphocytes. The frequency of alloimmunization was high (73%). Not only HLA antibodies, but also blood-cell-specific alloantibodies were detected in the sera of the alloimmunized patients, e.g. lymphocyte-specific, platelet-specific and granulocyte-specific antibodies.
The immunofluorescence test on platelets was also used as a crossmatch, and when this test was positive it was always found that after platelet transfusion the increment value was nil.     

Studies on levamisole--induced agranulocytosis

Widespread clinical trials of leavo-tetramisole (levamisole) as an immunopotentiating agent in rheumatoid arthritis, metastatic carcinoma, and immunodeficiency states have been complicated by agranulocytosis (AGC) in 2.5%-13% of patients. Other than a relationship with prolonged high dosage, very little is known regarding the pathogenesis of levamisole-induced AGC. Whereas leukoagglutination was negative, fluorochromatic microgranulocytotoxicity (GCY) tests were positive with serum from 10 of 10 acutely neutropenic patients. The antibody was IgM, reacted with 100% of unrelated granulocytes, but not with T or B lymphocytes. Some sera also reacted with monocytes and the myeloid cell line, K-562. Tests for antigen-antibody complexes or cold autoantibodies were negative. Although clinical evidence strongly suggests a haptene (drug) mechanism, in vitro mixing experiments were also negative. An alternative choice parallels the model of aldomet- induced Coombs'-positive hemolytic anemia. Finally, GCY first became positive 2-3 mo prior to the onset of AGC on two patients, suggesting the possibility of identifying those at risk well before the onset of neutropenia.     

K-cell lymphocytosis/neutropenia syndrome: the neutropenia is not caused by autoimmunity

The role of humoral immune mechanisms in the pathogenesis of the neutropenia in five patients with chronic killer (K)-cell lymphocytosis was studied. For the detection of neutrophil antibodies in the patient's blood, immunofluorescence, cytotoxicity and agglutination tests and a sensitive antibody-dependent cellular cytotoxicity (ADCC) assay were applied. An assay for bone-marrow colony growth (CFU-GM, BFU-E) inhibition was applied as well. A C1q-binding test was used to detect immune complexes. The lymphocytes of all five patients had the capacity to lyse alloantibody sensitized neutrophils. Neutrophil-bound IgG was found only in one patient. Two patients had neutrophil-reactive antibodies in their serum; in one patient these antibodies were only detectable in the ADCC using the patient's serum to sensitize target cells. However, these antibodies reacted also with lymphocytes, were absorbable with platelets and thus probably were HLA antibodies. The serum of one of these two patients showed complement-dependent CFU-GM and BFU-E inhibition, but the observed inhibition was probably also due to the anti-HLA antibodies present in his serum, as the inhibiting effect disappeared after absorption of the serum with platelets. The serum of only one patient contained low amounts of immune complexes, as measured in the C1q-binding test. Our data suggest that humoral autoimmune mechanisms, such as autoantibodies against neutrophils or neutrophil precursors or circulating immune complexes, do not seem to play an important role in the K-cell lymphocytosis/neutropenia syndrome. The possible role of the expanded K-cell population, and its humoral products (tumour necrosis factor, interferons), in the syndrome is discussed.     

The Evans syndrome: characterization of the responsible autoantibodies

The immunofluorescence test on paraformaldehyde-fixed cells was used for the detection of antibodies bound to the platelets and granulocytes and present in the sera of 24 patients with Evans syndrome and a further 29 patients with both idiopathic thrombocytopenia (ITP) and idiopathic neutropenia (INP), but without autoimmune haemolytic anaemia (AIHA). The direct immunofluorescence test on platelets and/or on granulocytes was positive in all patients with a cytopenia, but the sera of only 17 patients with the Evans syndrome and 15 of the other patients contained platelet- or granulocyte-specific autoantibodies.
From absorption and elution experiments, it appeared that the autoantibodies were directed against antigens specific for the various peripheral blood cells, i.e. erythrocytes, platelets and granulocytes and that they were not cross-reacting.     

Prospective Evaluation of the Chemiluminescence Test for the Detection of Granulocyte Antibodies: Comparison with the Granulocyte Immunofluorescence Test

A series of 213 neutropenic patients were tested for the presence of granulocyte antibodies using the granulocyte chemiluminescence test (GCLT) and the granulocyte immunofluorescence test (GIFT). Sera containing lymphocyte (HLA) antibodies were excluded from the study. A direct GIFT was performed on granulocytes from 56 patients. Samples were obtained from patients with a range of clinical conditions including primary adult autoimmune neutropenia, autoimmune neutropenia of infancy, autoimmune neutropenia secondary to Felty's syndrome, rheumatoid arthritis, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, proliferative disorders, bone marrow transplantation and patients with documented febrile or pulmonary transfusion reactions. Overall, granulocyte antibodies were detected in 52.1% of patient sera. Results for the GCLT and GIFT (IgG) were strongly correlated (plt;0.001) for both primary and secondary immune neutropenias. The results confirm the applicability of the GCLT in the granulocyte serology laboratory.     

Increased levels of soluble flt-3 ligand in serum and long-term bone marrow culture supernatants in patients with chronic idiopathic neutropenia

The levels of serum and long-term bone marrow culture supernatant soluble flt-3 ligand (sFL) were determined in 54 patients with chronic idiopathic neutropenia (CIN) and 16 normal controls. Both serum and supernatant sFL levels were significantly increased in the patients compared with controls. Individual sFL values inversely correlated with the number of circulating neutrophils and the proportion of bone marrow CD34+ cells. Supernatant sFL values positively correlated with the levels of supernatant G-CSF. These findings suggest that the impaired myelopoiesis in CIN patients is accompanied by a compensatory mechanism attempting to increase the neutrophil production at the myeloid progenitor cell level.     

Autoimmune Granulocytopenia: the Detection of Granulocyte Autoantibodies with the Immunofluorescence Test

Neutropenia may be caused by neutrophil autoantibodies. The detection of such antibodies has always been difficult. Recently, we developed a sensitive indirect immunofluorescence technique applicable to granulocytes which proved to be of value in the detection of granulocyte alloantibodies. We have now used this method to investigate the serum and cells of 29 patients with idiopathic or secondary neutropenia. In four patients neutrophil-antigen-specific autoantibodies were detected in the serum and the patient's own granulocytes showed direct fluorescence. Furthermore, differences in the immunoglobulin composition and the temperature of optimal activity of the autoantibodies were found. Direct fluorescence was also demonstrated with the granulocytes from five other neutropenic patients and with the granulocytes from five non-neutropenic patients mainly with infectious disease. However, no granulocyte antibodies could be eluted or shown to be present in the serum. This indicates that a positive direct granulocyte fluorescence test may not be considered as proof that autoantibodies are present.     

Investigating severe neutropenia with a simple bone marrow immunofluorescence test

Severely neutropenic patients who are suspected of having circulating autoantibodies to neutrophils and/or myeloid precursors present a diagnostic problem. This report describes the use of a simple bone marrow immunofluorescence test (BMIFT) to demonstrate autoantibodies reactive with the patient's own bands, neutrophils and myeloid precursors represented on fresh-frozen bone marrow aspirate smears. Positive results were seen in two of 13 evaluable marrows from children with primary neutropenia and in eight of 44 evaluable marrows from adults with primary or secondary neutropenia. The BMIFT is simple enough to perform in most laboratory environments and provides serological information to support a diagnosis of immune neutropenia when other means of investigation are precluded.     

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.     

Disseminated disease due to Mycobacterium chelonei treated with amikacin and cefoxitin. Absence of killing with either agent and possible role of granulocytes in clinical response

Disseminated disease due to rapidly growing mycobacteria is manifested by positive blood cultures and multiple skin and subcutaneous abscesses. A patient had T-cell lymphoma and disseminated disease; he also had neutropenia intermittently. Single-agent therapy with amikacin sulfate or cefoxitin sodium was not adequate during periods of neutropenia, and combination therapy was necessary to control the infection. Clinical response correlated with detectable serum inhibitory levels of the antimicrobial agents. Surprisingly, the organism was not killed by either amikacin or cefoxitin, a finding that correlated with the absence of serum bactericidal levels. This case suggests that granulocytes may play a role in the host's response to this organism, and determination of serum inhibitory and possible bactericidal levels may be useful in monitoring therapy.     

Serum levels of lactoferrin and myeloperoxidase in chronic idiopathic and secondary neutropenia

In 20 patients with chronic neutropenia, serum lactoferrin (S-LF) and serum myeloperoxidase (S-MPO) levels were assessed. By immunofluorescence, granulocyte-bound immunoglobulins were detected in 12 patients, whereas circulating immune complexes were found in the blood of 8 patients by the ¹²⁵-I-Clq-binding test (Clq-BT). In both groups of patients, there was a relative increase of S-LF and a relative or sometimes absolute increase of S-MPO. In the latter group, results of the Clq-BT correlated positively with S-MPO but negatively with neutrophil counts. No correlations between S-LF or S-MPO and the results of the granulocyte immunofluorescence test were found. Our results suggest that S-LF and S-MPO levels may be helpful in the further study of patients with chronic neutropenia, to gain more insight into the pathogenetic mechanisms operative in this disease.     

Serum levels of lactoferrin and myeloperoxidase in chronic idiopathic and secondary neutropenia. A preliminary report

In 20 patients with chronic neutropenia, serum lactoferrin (S-LF) and serum myeloperoxidase (S-MPO) levels were assessed. By immunofluorescence, granulocyte-bound immunoglobulins were detected in 12 patients, whereas circulating immune complexes were found in the blood of 8 patients by the 125-I-C1q-binding test (C1q-BT). In both groups of patients, there was a relative increase of S-LF and a relative or sometimes absolute increase of S-MPO. In the latter group, results of the C1q-BT correlated positively with S-MPO but negatively with neutrophil counts. No correlations between S-LF or S-MPO and the results of the granulocyte immunofluorescence test were found. Our results suggest that S-LF and S-MPO levels may be helpful in the further study of patients with chronic neutropenia, to gain more insight into the pathogenetic mechanisms operative in this disease.     

Discrepancy between Clq deviation and Raji cell tests in detection of circulating immune complexes in patients with leprosy.

Samples of serum from 45 patients with different clinical forms of leprosy and from 17 patients with systemic lupus erythematosus were studied in parallel for circulating immune complexes with use of two different in vitro tests adjusted to the same degree of sensitivity. The Clq deviation test relied upon the reaction of the complement component Clq with immune complexes. The Raji cell test detected complement-fixed immune complexes that bound to the complement receptors on cultured, bone marrow-derived lymphocyte-like Raji cells. Thirty (67%) of 45 patients with leprosy showed immune complexes according to the Clq deviation test; however, only two (7%) of the 30 samples of sera with positive Clq test results were positive by the Raji cell test. In contrast, 54% of 13 samples of sera from patients with systemic lupus erythematosus positive by the Clq test were positive according to the Raji cell test. Since Clq is known to react with DNA as well as with bacterial antigens, the Clq reaction may in fact be detecting antigenemia in many instances. Considerable caution is warranted in application of sensitive screening tests for assay of circulating immune complexes in various states of infectious diseases.     

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.     

Characterization of the bone marrow immunofluorescence test in childhood autoimmune neutropenia

The bone marrow immunofluorescenece test (BMIFT) demonstrates autoantibodies to granulocytes and their precursors on fresh-frozen bone marrow slides. It may be used to differentiate childhood autoimmune neutropenia (AIN) from other causes of childhood neutropenia, even when circulating neutrophil counts are low. We sought to characterize the diagnostic utility of the BMIFT in childhood AIN. All BMIFT requests for investigation of children with neutropenia between January 1998 and May 2007 were reviewed. Patients were classified as AIN or nonautoimmune causes. Baseline demographic data, results of BMIFT, granulocyte immunofluorescence testing and bone marrow findings were collected from clinical records and the institutional laboratory database. Seventy-six children had BMIFT performed for investigation of neutropenia. There were 45 patients diagnosed with AIN, 28 with nonimmune neutropenia and three failed tests. The median age of children with AIN was 1.2 years (range 0.3-15.3), compared with 3.6 years (range 0.1-15.7) in the nonautoimmune group. The median neutrophil count in AIN was 0.3 x 10(9)/l (0.9 x 10(9)/l in nonautoimmune). BMIFT was positive in 24 of 45 patients with AIN and 0 of 28 with nonautoimmune neutropenia (sensitivity 53%, specificity 100%, positive predictive value (PPV) 100%, negative predictive value 57%). Ten patients had other autoimmune diatheses at diagnosis. The BMIFT is a simple, highly specific test with excellent PPV and thus is a clinically useful test to confirm AIN in children.     

Expression of Ia-antigens on normal and chronic myeloid leukemic human granulocyte-macrophage colony-forming cells (CFU-GM) is associated with the regulation of cell proliferation by prostaglandin E

The presence of la-antigens and their relationship to the inhibitory effect of prostaglandin E on the proliferation of human CFU-GM was studied in animals and patients with chronic myeloid leukemia. Consistent reduction of normal colony formation to approximately 50% of baseline levels was observed using a monoclonal anti-human la antibody in a complement-dependent cytotoxicity assay titrated over serial dilutions. ELimination of the la-antigen-bearing CFU-GM population was associated with virtually a complete loss of responsiveness to the inhibitory effects of prostaglandin E. Maintenance of bone marrow cells in short-term suspension culture at 37 degrees C prior to agar culture resulted in the loss of detectable la-antigen on the CFU-GM and, similarly, loss of response to prostaglandin. In contrast, most patients with chronic myeloid leukemia showed greatly reduced levels of la-antigens on their CFU-GM in fresh marrow together with lack of prostaglandin sensitivity, suggesting a correlation with the abnormal growth regulation observed in these patients. In two chronic myeloid leukemia patients, levels of la-antigen higher than that observed in the majority of patients could be detected and correlated with a residual response to prostaglandin E. These results suggest a relationship in normals between the expression of la-antigens on CFU-GM and the physiologic response to regulation by prostaglandin E, and a possible mechanism for the aberrant regulatory response in patients with chronic myeloid leukemia.     

Antibody-dependent lymphocyte-mediated granulocyte cytotoxicity in man

Sera from two patients with granulocytopenia associated with collagen vascular disease caused the destruction of normal human granulocytes by autologous lymphocytes in vitro. Granulocyte cytotoxicity was measured by the release of 51Cr during incubation with test sera and lymphocytes in microtiter plates. Between 8% and 46% granulocytoxicity was produced in granulocytes from 8 normal donors by the sera from these two patients. Less than 6% granulocytotoxicity was seen with the sera from 14 normal subjects and 29 patient controls. Treatment of lymphocyte preparations with carbonyl iron and magnetic separation to remove phagocytic cells or treatment with complement-coated red cells followed by repeated gradient centrifugation to remove complement receptor- bearing lymphocytes did not reduce the granulocytotoxicity. There was a dose-response relationship between the concentration of positive sera and granulocytotoxicity. When these sera were fractionated by Sephadex G-200 gel filtration and by ion-exchange chromatography with DEAE- cellulose, the active component appeared in the IgG-containing fractions. Thus, IgG antibody-dependent, lymphocyte-mediated granulocyte cytotoxicity represents a means of detecting human granulocyte antibodies and is a possible mechanism of autoimmune neutropenia in these two patients.