Faculty Bibliography

The medical records of patients receiving cyclophosphamide for lupus nephritis between 1987 and 1993 at the New York University/Hospital for Joint Diseases Lupus Study Group Institutions were retrospectively reviewed. We identified 45 patients (38 female, seven male) who received a mean of 9 +/- 1 (range 2-23) pulses of intravenous cyclophosphamide for diffuse proliferative glomerulonephritis (n = 28), focal proliferative glomerulonephritis (n = 7), membranous nephropathy (n = 5), mesangial nephropathy with sclerosis (n = 1) or nephritis without biopsy (n = 4). Forty-two of the 45 patients received cyclophosphamide after failing steroid therapy. During a follow-up period of 52 +/- 3 months, nine patients progressed to end-stage renal disease (ESRD) with three additional patients experiencing a doubling of the creatinine and two patients persistent nephrotic range proteinuria. There were no deaths directly attributable to cyclophosphamide and no patients developed hemorrhagic cystitis or malignancy. Ten of 37 women had ceased menstruating prior to cyclophosphamide therapy. Treatment-associated amenorrhea occurred in only three patients all over 27 years of age. Intermittent intravenous cyclophosphamide therapy of lupus nephritis is well tolerated and usually effective in maintaining renal function in patients unresponsive to steroids although, in our experience, 20% of patients developed ESRD and a total of 14 of 45 (30%) patients had unsatisfactory outcomes

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically

It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show 'constitutively expressed' iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells

In these studies we provide conclusive evidence that (beta/gamma) actin present in human neutrophils is a substrate for nitric oxide (NO)-dependent ADP ribosylation and that this modification is associated with the inhibition of actin polymerization. A 43-kDa substrate for NO-dependent ADP ribosylation was identified as actin by four methods: (1) comigration with the botulinum C2 toxin substrate by two-dimensional gel electrophoresis (pI 5.2), (2) identity between the peptide map generated by V8 protease digestion of the NO and botulinum C2 substrates, (3) immunoprecipitation with antiactin antibodies, and (4) the ability of NO to ADP ribosylate purified neutrophil G-actin in the presence of plasma membrane cofactors. Because the ADP ribosylation of actin by the botulinum C2 toxin is known to inhibit F-actin polymerization, we examined the effect of NO on actin assembly. Flow cytometry revealed that NO inhibited formyl-methionine-leucine-phenylalanine (fMLP)-dependent (30 s at 37 degrees C) F-actin formation (108 +/- 8 vs. 89 +/- 6 relative fluorescence units, P < .02). These results were confirmed by quantification of F-actin formation by gel scanning (10% sodium dodecyl sulfate gel, Coomassie, and densitometry): pretreatment of polymorphonuclear leukocytes with NO resulted in a reduction of fMLP-induced, cytoskeletal-associated F-actin, which was accompanied by an increase of Triton-soluble G-actin. NO also inhibited F-actin formation, as observed by means of rhodamine phalloidin staining of neutrophils adherent to a fibronectin-coated surface. This effect was accompanied by a dose-dependent inhibition of neutrophil adherence in NO-treated cells. The data indicate that NO inhibits cytoskeletal assembly and adherence in human neutrophils in association with the ADP ribosylation of actin

The destructive capacity of the neutrophil has long been appreciated, and the presence of extraordinary numbers of neutrophils in the synovial fluid of patients with RA supports a role for these cells in the pathogenesis of joint destruction. In this article, we reviewed the current state of knowledge of neutrophil function in the inflammatory response, and emphasized the subjects of neutrophil/endothelial adhesion and the role of chemoattractants and cytokines in neutrophil mobilization. We also discussed the mechanisms of action of neutrophil destruction of cartilage and the interplay of signals between the neutrophil and the chondrocyte. The capacity of many of the drugs used to treat RA to interfere with one or several of these processes underscores the importance of the neutrophil in RA and suggests that future therapeutic strategies could target neutrophil activation within the synovial space