Faculty Bibliography

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C]glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity

We sought to determine whether the activation event which renders the CD11/CD18 leukocyte integrin/Leu-CAM glycoproteins capable of promoting cell to cell adhesion was associated with the induced posttranslational modification of phosphorylation. In neutrophils, two species of alpha-chains, a predominant CD11b 165-kDa subunit and a minor 150-kDa CD11c subunit were found to be constitutively phosphorylated. However, the 95-kDa CD18 common beta-chain was not phosphorylated in resting cells but became strongly phosphorylated in cells incubated with PMA. The beta-chain was phosphorylated in a dose-related manner within 1 min of the addition of PMA, reached maximal intensity between 4 to 10 min, and remained fully phosphorylated at 30 min. The similarities of the kinetics of homotypic aggregation induced by PMA to the time course of phosphorylation suggest that phosporylation may be relevant to at least this type of Leu-CAM-dependent adhesion. In contrast, in the presence of FMLP which induces aggregation with different kinetics than PMA, no phosphorylation of the common beta-chain was observed over a time interval of from 30 s to 10 min further emphasizing the apparent differences in the two modes of activation to an adhesive state. The phosphorylated species on neutrophils were readily detected by immunoprecipitation with each CD18 mAb and most but not all CD11b mAb which otherwise precipitated 125I-labeled CD11b species suggesting that the CD11b alpha-chain labelled with 32P may differ slightly from the 125I-labeled species in terms of its recognition by certain CD11b mAb. In mononuclear cells, similar constitutive phosphorylation of the CD11a and CD11c alpha-chains was observed that remained unchanged in the presence of either FMLP or PMA. As was demonstrated in neutrophils, phosphorylation of the CD18 beta-chains of mononuclear cells was not constitutive but was induced in the presence of PMA and not FMLP. Taken together these data suggest the existence of specific recognition sites on beta-chains for a regulatory kinase-phosphatase system

The iC3b receptor (CR3) is required for neutrophil adhesive functions, including homotypic aggregation. Because stimuli that enhance neutrophil adhesion also induce up-regulation of surface CR3, it is widely held that these two responses are causally related. We have dissociated CR3 display (immunofluorescence) from CR3 function (aggregation). Neutrophils isolated at 4 degrees C and rewarmed to 37 degrees C up-regulated surface CR3 twofold, but did not aggregate. The kinetics of FMLP-induced CR3 up-regulation were discordant with those of aggregation. In the absence of extracellular divalent cations, CR3 expression increased twofold after exposure to FMLP, but neutrophils did not aggregate. FMLP elicited 3.5-fold more aggregation than the ionophore A23187, yet less than one-half as much CR3 up-regulation. 3 mM sodium salicylate inhibited aggregation 55 +/- 4%, but had no effect on CR3 up-regulation. Conversely, 1 mM tetracaine completely inhibited CR3 up-regulation, while significantly enhancing aggregation. Neutroplasts expressed CR3, but did not up-regulate the receptor; in contrast, FMLP induced CR3-dependent aggregation of neutroplasts. We conclude that, although constitutive surface CR3 is required for neutrophil aggregation, the up-regulation of CR3 is neither necessary nor sufficient to promote cell-cell adhesion

Whether homotypic neutrophil aggregation depends on the quantitative increase of gp165/95 molecules (Mac 1, CR3) recruited to the cell surface during activation was studied using mAb of the CD11b group that recognize distinct epitopes encoded by the alpha-subunit of this glycoprotein. After the addition of antibody MN41, neutrophils did not aggregate in response to a chemoattractant, FMLP. Blockade of preexisting surface gp165/95 by mAb MN41, followed by removal of the excess antibody from the mixture, was used to show that the molecules of gp165/95 newly expressed in response to stimulation by a chemoattractant were incapable of effectively mediating the induced cell-cell interactions of aggregation. Flow cytometry studies confirmed that binding of unlabeled antibody MN41 did not block further increases in surface expression of gp165/95 after stimulation with FMLP. These data suggest that molecules of gp165/95 exhibit two functionally distinct forms, one, present on the surface of freshly isolated neutrophils, that becomes competent to mediate the aggregation response upon activation by a stimulus and a second form that can be translocated to the cell surface by the stimulus but is greatly diminished if not lacking in the ability to participate in that aggregation event

We measured levels of complement anaphylatoxin split products, C3a and C5a, in the circulation of patients with systemic lupus erythematosus (SLE). In 23 SLE patients who were followed serially, the mean C3a value was 179 ng/ml during stable disease and 550 ng/ml during a disease flare. In 10 patients, C3a levels predicted disease activity, with the C3a value rising from a mean of 183 ng/ml at a time of stable disease to a mean of 242 ng/ml 1-2 months prior to a clinical exacerbation of disease. The mean C3a level in 5 patients with acute dysfunction of the central nervous system (CNS) was 1,297 ng/ml, which is significantly higher than that observed in patients with active disease but without CNS involvement (P less than 0.01). C5a levels were also significantly elevated in 4 patients with acute CNS disease. Pathologic specimens from 2 patients who died during an acute lupus flare revealed neutrophils occluding the cerebral and intestinal vessels. Fluorescein angiography in a patient with CNS lupus revealed vasoocclusive retinopathy. In 5 of 7 SLE patients who were pregnant, C3a levels were elevated, with a group mean value of 310 ng/ml. There was a negative correlation (r = -0.59) between C3a and C3 levels in pregnant patients with SLE, and this finding is consistent with complement activation as the cause of decreasing C3 levels. We suggest that serial measurements of C3a can predict flares of disease in lupus patients and can demonstrate complement activation during pregnancy in women with SLE. In addition, release of C3a and C5a (mediators of inflammation) into the circulation may elicit vascular injury, particularly in patients with lupus cerebritis