Neutrophil-binding immunoglobulin G in systemic lupus erythematosus.

The objectives of these studies were to quantify the amounts of immunoglobulin (Ig)G bound to peripheral blood neutrophils from patients with systemic lupus erythematosus (SLE) and to determine the contributions of soluble immune complexes or anticell antibodies to the levels of IgG neutrophil-binding activity in SLE sera. Neutrophil-bound IgG, determined by a sensitive antiglobulin inhibition assay, was elevated in 7 out of 14 SLE patients compared with values obtained in 23 normal controls. The levels of IgG neutrophil-binding activity in sera were elevated in 22 of 38 patients with SLE over the values seen with 36 normal sera. No correlation was found between the peripheral blood neutrophil counts in the SLE patients and the values for IgG adherent to the cells or serum cell-binding activity. The sera from 18 patients with SLE were fractionated by gel filtration. Elevated levels of IgG neutrophil-binding activity were found in 11 of the 18 G-200 excluded pools and in 13 of the G-200 IgG pools. In nine sera elevated levels were observed in both pools. F(ab')2 fragments of IgG from SLE sera bound to normal polymorphonuclear leukocytes in greater amounts than F(ab')2 fragments of IgG from normal sera. A significant correlation existed between the values of IgG neutrophil-binding activity found in SLE sera and those obtained with both the G-200 excluded and IgG pools. Sucrose density gradient fractionation of four sera from SLE patients confirmed the presence of both large (greater than 19S) and intermediate-sized (7S-19S) cell-binding immune complexes as well as of monomeric IgG antibodies to neutrophils. The levels of IgG neutrophil-binding activity in the SLE sera correlated well with the results obtained with the Raji cell assay for immune complexes as well as with the titer of antibodies to nuclear antigens. These data indicate that circulating neutrophils from patients with SLE commonly have increased amounts of cell-bound IGG. The elevated levels of IgG neutrophil-binding activity in the sera of these patients are caused by both soluble immune complexes and antibodies reactive with neutrophils.

Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine.

We have examined whether the toxic effects of homocysteine on cultured endothelial cells could result from the formation and action of hydrogen peroxide. In initial experiments with a cell-free system, micromolar amounts of copper were found to catalyze an oxygen-dependent oxidation of homocysteine. The molar ratio of homocysteine oxidized to oxygen consumed was approximately 4.0, which suggests that oxygen was reduced to water. The addition of catalase, however, decreased oxygen consumption by nearly one-half, which suggests that H2O2 was formed during the reaction. Confirming this hypothesis, H2O2 formation was detected using the horseradish peroxidase-dependent oxidation of fluorescent scopoletin. Ceruloplasmin was also found to catalyze oxidation of homocysteine and generation of H2O2 in molar amounts equivalent to copper sulfate. Finally, homocysteine oxidation was catalyzed by normal human serum in a concentration-dependent manner. Using cultured human and bovine endothelial cells, we found that homocysteine plus copper could lyse the cells in a dose-dependent manner, an effect that was completely prevented by catalase. Homocystine plus copper was not toxic to the cells. Specific injury to endothelial cells was seen only after 4 h of incubation with homocysteine plus copper. Confirming the biochemical studies, ceruloplasmin was also found to be equivalent to Cu++ in its ability to cause injury to endothelial cells in the presence of homocysteine. Since elevated levels of homocysteine have been implicated in premature development of atherosclerosis, these findings may be relevant to the mechanism of some types of chronic vascular injury.

The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus.

To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B)-DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B)-DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B)-DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin.

Thromboangiitis obliterans associated with idiopathic hypereosinophilia.

A 47-year-old male smoker with chronic eosinophilia developed progressive ischemia in his extremities. Pathologic examination of vessels in amputated limbs revealed changes of thromboangiitis obliterans. Focal segments of some arteries, however, revealed infiltration of thrombus and vessel wall by eosinophils. Furthermore, the patient developed a totally occluded right temporal artery, which on biopsy specimen showed marked infiltration by eosinophils within the vessel wall. In contrast to most patients with the hypereosinophilic syndrome, however, this patient had no evidence of endomyocardial thrombosis or fibrosis. In view of recent evidence that eosinophil granule proteins are toxic to endothelial cells, the findings in this patient suggest the possibility that eosinophils may be involved in the pathogenesis of thromboangiitis obliterans.

Suppression of in vitro granulocytopoiesis by captopril and penicillamine.

The mechanisms underlying drug-induced neutropenia are poorly characterized. We have examined the mechanism of suppression of granulocytopoiesis by captopril and penicillamine using human and canine bone marrow cells in an in vitro culture system. Addition of captopril caused no significant change in granulocyte-macrophage colony formation at concentrations up to 30 micrograms/ml. In the presence of CuSO4 (1-3 micrograms/ml), however, captopril caused significant inhibition of colony growth (p less than 0.05). Penicillamine, another agent associated with neutropenia and, like captopril, having a reactive thiol group, also inhibited colony formation in the presence of copper. Chemical congeners of captopril lacking a reactive thiol group and enalaprilic acid, an alternative angiotensin-converting enzyme (ACE) inhibitor, failed to show inhibition, suggesting that the thiol group and not ACE inhibition was responsible. Analysis of day-7 colonies (98% neutrophilic) and day-21 colonies (37% neutrophilic, 30% macrophagic, 27% eosinophilic, and 6% mixed) showed that neutrophil-containing colonies, but not nonneutrophilic colonies were inhibited by the addition of captopril plus copper. Catalase totally reversed the inhibition of colony formation caused by these agents. Direct measurement of oxygen consumption in the presence of captopril showed marked enhancement with the addition of CuSO4 and a 48% reduction in the presence of added catalase. These data indicate that drugs with a reactive thiol group can interact with copper to generate H2O2, which can be toxic to neutrophilic progenitor cells. We postulate that this may be an important mechanism for drug-associated neutropenia and a general mechanism for drug-induced marrow cell injury.

Large granular lymphocyte leukemia. Report of 38 cases and review of the literature.

LGL leukemia results from a chronic, clonal proliferation of LGL. Chronic neutropenia with recurrent bacterial infection and splenomegaly are common clinical manifestations. Rheumatoid arthritis coexists in some of these patients, who thus resemble patients with Felty syndrome. Other hematologic abnormalities that may occur include pure red-cell aplasia and adult-onset cyclic neutropenia. Lymphoid infiltration of bone marrow, splenic red pulp cords, and hepatic sinusoids is characteristic; lymph node and skin involvement are rare. Multiple serologic abnormalities are frequently present, including positive tests for rheumatoid factor and/or antinuclear antibody, polyclonal hypergammaglobulinemia, and circulating immune complexes. Antineutrophil and antiplatelet antibodies are often present. Leukemic LGL exhibit phenotypic heterogeneity; the most common phenotype in our patients is CD2+, CD3+, CD8+, HNK-1+, CD16-. Despite markedly increased numbers of LGL, functional activity of the cells is usually decreased. The mechanism of cytopenias is uncertain: in pure red-cell aplasia, it appears to be due to suppressive effect on erythropoiesis by abnormal LGL, but in patients with chronic neutropenia it may be antibody-mediated. Although most patients appear to have a relatively benign clinical course, mortality from infections and progressive lymphoproliferation is substantial. Optimal therapy remains undefined. Some preliminary evidence suggests that LGL leukemia may be associated with infection with a retrovirus similar to HTLV-I. Although relatively rare, LGL leukemia is of interest because a better understanding of this disease process may contribute to our knowledge of autoimmune diseases, the immunoregulatory functions of LGL, and the mechanisms controlling normal hematopoiesis.

Prototypical HTLV-I/II infection is rare in LGL leukemia.

The etiology of LGL leukemia is not known; however, we recently detected HTLV-II in a patient with LGL leukemia. In this study, we found that sera from 6 of 28 patients with LGL leukemia were positive for HTLV-I/II using a whole virus ELISA; moreover, the ELISA-negative sera were near the positive cut-off value. Therefore, we performed additional studies on these sera using commercially available assays which can confirm and distinguish HTLV-I from HTLV-II infection. Serum from only one patient was confirmed positive using conventional criteria (HTLV-II+). Sera from 25 patients (89%) had indeterminate reactivity on Western blot assays. Of these, sera from 21 (84%) reacted to gag protein p24; 12 (48%) reacted with recombinant env protein p21e, and 10 (40%) reacted with both. We could not detect HTLV-I/II pol or pX gene sequences in these patients using polymerase chain reaction analyses, with the exception of the HTLV-II-infected patient described previously. These data show that most patients with LGL leukemia are not infected with prototypical HTLV-I or HTLV-II. The frequent reactivity of patient sera to HTLV-I/II gag protein p24 and to env protein p21e, however, suggests that a deleted or variant form of HTLV-I/II may be associated with LGL leukemia.

Pancytopenia associated with low dose pulse methotrexate in the treatment of rheumatoid arthritis.

Low dose pulse MTX was associated with the development of pancytopenia in six patients with RA. Two patients died. Factors implicated in the occurrence of this complication were renal impairment in five patients, medication errors by two patients, preexisting marrow injury from occult alcoholism in one patient, and an apparent idiosyncratic reaction to the drug in another. Medication errors were associated with the use of five or more medications, and the unusual schedule of administration of low dose MTX may also have been contributory. From a consideration of the clinical pharmacokinetics of MTX, we suggest other factors that may predispose to the occurrence of marrow toxicity: the presence of hypoalbuminemia, interactions between MTX and other protein bound or weakly acidic drugs, and the repetitive dosing schedule of low dose MTX. Based on our experience, patients with impaired renal function (creatinine greater than or equal to 2.0 mg/dL) should not receive MTX. Renal function should be monitored regularly during treatment with MTX, and blood counts should be observed carefully if a new drug is added or substituted. A 5 mg test dose of MTX before initiating weekly therapy may identify patients with severe hypersensitivity to the drug. The potential risks of using MTX in a patient unwilling to accept blood products should be acknowledged and discussed with the patient. Furthermore, we recommend the use of leucovorin if pancytopenia occurs, even if low or undetectable serum levels of MTX are present.

The pathology of large granular lymphocyte leukemia.

The authors conducted a histopathologic study on tissues from 11 patients with the recently described syndrome of large granular lymphocyte (LGL) leukemia. Distinctive pathologic findings were found most often in the bone marrow, liver, and spleen. The bone marrow biopsies contained nonparatrabecular lymphoid infiltrates that were nodular or diffuse and interstitial. Plasmacytosis was found and in two cases there was myeloid maturation arrest. The liver biopsies contained sinusoidal and portal infiltrates and the spleen had red pulp cord and sinus infiltrates, plasmacytosis, and follicular hyperplasia. Lymph node involvement was nondiagnostic, consistent with the usual absence of lymphadenopathy. The morphologic findings were sometimes indistinguishable from other reactive or low-grade lymphoproliferative disorders, especially chronic lymphocytic leukemia/well-differentiated lymphocytic lymphoma (CLL/WDLL) and hairy cell leukemia. These results suggest the need to correlate peripheral blood cell counts and morphology as well as immunophenotypic studies with tissue histology to distinguish LGL leukemia from other disorders. Establishing a correct diagnosis of LGL leukemia may help clarify the etiology of unexplained peripheral blood cytopenias, arthritis, and other autoimmune manifestations in individual patients.

Humoral immune abnormalities in T-cell large granular lymphocyte leukemia.

The prevalence of humoral immune dysfunction has not been defined in a large series of patients with T-cell large granular lymphocyte leukemia (T-LGL) confirmed to be clonal by T-cell receptor analysis. Therefore we evaluated the presence of multiple autoantibodies in 27 patients with this disease. Humoral immune abnormalities included: rheumatoid factor (RF) (15/27 patients), antinuclear antibody (ANA) (13/27 patients), polyclonal hypergammaglobulinemia (15/24 patients), elevated serum immunoglobulins (17/26 patients), immune complex formation (18/25 patients), elevated beta-2 microglobulin (13/18 patients) and neutrophil-reactive IgG (18/20 patients). Disease manifestations in these patients were due to complications of cytopenia or autoimmune abnormalities. Infection was a common finding (21/27 patients) and likely reflected their neutropenia. Rheumatoid arthritis (11/27 patients), anemia (12/27 patients) and thrombocytopenia (10/27 patients) were less common but still frequently observed. This study demonstrates the presence of multiple autoantibodies in a large series of patients with documented clonal T-LGL proliferations.

Use of colony-stimulating factors in the treatment of neutropenia associated with collagen vascular disease.

Chronic neutropenia associated with collagen vascular disease is seen principally with Felty's syndrome complicating rheumatoid arthritis. Multiple recent reports document the efficacy of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in reversing the neutropenia and decreasing the risk of infections in Felty's syndrome. Long-term use of G-CSF appears well tolerated and effective in Felty's syndrome. Of concern, however, have been flares of arthritis and development of leukocytoclastic vasculitis in several patients following the use of colony-stimulating factors (CSFs) in Felty's syndrome. The incidence of these complications of CSF therapy appears to be greater in Felty's syndrome than in other disorders. Future studies will need to address the incidence of these side effects, evaluate strategies to reduce risks, and clarify the optimum use of CFSs in Felty's syndrome.

Monocyte Fc receptor function in rheumatoid arthritis. Enhanced cell-binding of IgG induced by rheumatoid factors.

Monocytes from 11 patients with rheumatoid arthritis and 10 control subjects were purified by countercurrent elutriation. Rheumatoid arthritis monocytes had more cell-associated IgG (P less than 0.001) and bound more 125I-labeled heat-aggregated IgG in vitro (P less than 0.02) than did monocytes from control subjects. Interaction of rheumatoid factor (RF) with monocytes was then investigated. Purified 125I-labeled IgM-RF and IgG-RF bound directly to monocytes from normal individuals. Furthermore, preincubation of normal monocytes with RF augmented subsequent binding of aggregated IgG to the cells. We conclude that monocyte-associated RF can enhance binding of IgG-containing immune complexes to the cells and can exaggerate the measured number of Fc receptors. Such cell-bound RF may affect clearance of immune complexes by the reticuloendothelial system in vivo.

D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation.

Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.

Pasteurella pneumonia: report of a case and review of the literature.

A case of pneumonia due to Pasturella ureae was encountered in a 57-year-old man who developed bilateral pulmonary infiltrates and respiratory insufficiency while convalescing in the hospital from a hip fracture and multiple rib fractures. Cultures of his sputum grew an essentially pure growth of Pasturella ureae. This organism, a small gram-negative rod, could be differentiated from the other Pasteurella species by its ability to hydrolyze urea and failure to produce indole. The literature on Pasteurella infections is briefly reviewed, and the recent taxonomic revisions of the genus Pasteurella are discussed.

Colchicine induced rhabdomyolysis.

A 59-year-old man with chronic obstructive pulmonary disease (COPD), atrial fibrillation, and gout developed acute dyspnea, cough, and diffuse muscle aches and pains. He had commenced colchicine (0.6 mg b.i.d. p.o.), for the first time, one month earlier for recurrent gout attacks. Clinical examination revealed atrial fibrillation, an exacerbation of his pulmonary disease, tender muscles, especially calves, and diffuse muscle weakness. Laboratory results included creatinine phosphokinase 6961 U/l (1% MB), microscopic hematuria, myoglobinuria, elevated creatinine 1.6 mg/dl, and blood urea nitrogen 17 mg/dl. COPD and atrial fibrillation were treated and colchicine was discontinued. The patient made a full recovery. This 2nd reported case of colchicine induced rhabdomyolysis is the first reported in the treatment of gout.

Rheumatic disorders associated with hepatitis C.

The hepatitis C virus (HCV) is a common virus of world-wide distribution affecting up to 3% of the world's population. Its genetic diversity, with multiple subtypes, and existence in the form of quasispecies in individual hosts, is, in part, responsible for high rates of chronic infection. Individuals with HCV infection will undoubtedly present to rheumatologists and other health care professionals with rheumatic and other immunological disorders related to what was usually a remote and asymptomatic acute infection. The goals of this review are: (1) to summarize clinical observations regarding rheumatological and immunological diseases linked with HCV infection; (2) to provide relevant information on the molecular biology of HCV; (3) to discuss the state of the art regarding the use of diagnostic studies; (4) to consider the differential diagnosis of liver disease and rheumatic disorders; and (5) to provide a practical guide to the history, physical examination, laboratory work-up, disease monitoring, and therapy of HCV patients with rheumatic disorders.

Isolated central nervous system vasculitis associated with hepatitis C infection.

Since its identification in 1989, hepatitis C has been implicated in the pathogenesis of an increasing number of diseases previously believed to be primary or idiopathic. We report 2 rarely seen cases of isolated central nervous system (CNS) vasculitis in patients with hepatitis C infection. Patient 1. A 43-year-old man with 4 day right temporal headache developed a left hemiparesis. Weakness was his only physical finding. Computed tomography (CT) scan demonstrated a large right frontotemporal hemorrhage, and angiography revealed focal dilatations and irregularities of multiple branches of the right middle and anterior cerebral arteries. Cerebral decompression was performed and leptomeningeal biopsies showed granulomatous angiitis. Laboratory results were normal except for elevated liver biochemical tests. Later testing for hepatitis C was positive. His neurological symptoms improved with corticosteroids and cyclophosphamide. Patient 2. A 39 yr old male developed 3 days of left sided weakness, slurred speech and difficulty swallowing fluids. Physical findings were limited to his weakness. Magnetic resonance imaging demonstrated a right superior pontine subacute infarct with a small left internal capsule lacunar infarct. Angiography revealed multiple areas of focal narrowing with no areas of abrupt vessel cut off. Cerebral spinal fluid showed 71 PMN, 29 RBC, normal glucose, elevated protein (64 mg/dl), no oligoclonal bands, and low myelin basic protein. Other laboratory analyses were normal including liver biochemical tests. However, hepatitis C serology was positive and mixed cryoglobulins were detected. CNS vasculitis was diagnosed and nearly full recovery was achieved with corticosteroids, cyclophosphamide and warfarin.

Similar rearrangements of T-cell receptor beta gene in cell lines and uncultured cells from patients with large granular lymphocyte leukemia.

We established interleukin-2-(IL-2) dependent cell lines from three patients with large granular lymphocyte (LGL) leukemia. Phenotypic analysis demonstrated retention of the CD3+, CD8+ phenotype that was observed in the original leukemic LGL. Unique rearrangements of T-cell receptor beta gene occurring in uncultured leukemic LGL, were also found in cell lines, which suggests that the cell lines were derived from the original leukemic LGL clone in each case.

Clonal proliferation of large granular lymphocytes in rheumatoid arthritis.

Some patients with chronically elevated large granular lymphocyte (LGL) numbers have rheumatoid arthritis (RA). Since these patients also may have neutropenia and splenomegaly, their symptoms resemble those of patients diagnosed as having Felty's syndrome (FS). We studied the immunophenotypic and genotypic characteristics of mononuclear cells from patients with RA and neutropenia to better determine the extent of heterogeneity in this condition. Four patients had markedly increased numbers of LGLs, which expressed HNK-1 antigen and IgG Fc receptors. In contrast, the remaining 8 patients, who had FS, had normal LGL counts, and surface marker studies showed normal numbers of HNK-1 and IgG Fc receptor positive cells. Clonal rearrangement of the T cell receptor beta chain gene was demonstrated in all 4 patients with excess LGLs, whereas a germline configuration of this gene was present in all 6 FS patients in whom this was studied. These results suggest that there are diverse groups among patients with RA and neutropenia. Since prognosis may differ, it is important to recognize that some patients who are considered to have Felty's syndrome may have a clonal proliferation of LGLs.