Initial evaluation of an ultrasound measure for assessing the activity of skin lesions in juvenile localized scleroderma.

OBJECTIVE: To evaluate the construct validity of 2 proposed measures (the Ultrasound Disease Activity [U-DA] and the Tissue Thickness Score [TTS]) for evaluating sonographic differences in juvenile localized scleroderma skin lesions. METHODS: We conducted a retrospective review of juvenile localized scleroderma patients who had ultrasound scans of their skin lesions between October 2005 and February 2009. Imaged lesions were classified as active or inactive based upon clinical assessment. Lesions had to have been imaged within 1 month of a clinic visit or have the same clinical assessment during both the visit before and the visit after the scan. Two physicians scored the scans using the U-DA, which scores for differences in lesion echogenicity and vascularity compared with normal tissue. Tissue thickness differences were evaluated by percent differences and by using the TTS. Wilcoxon's rank sum test was performed to assess differences. RESULTS: We studied 52 scans from 21 patients, 32 scans of active skin lesions and 20 scans of inactive skin lesions. Features reported by clinicians as indicative of active disease included erythema, warmth, violaceous color, new lesion, expansion of lesion, and induration. The U-DA was significantly different between active and inactive skin lesions (P = 0.0010) with significant differences found for the parameters of total echogenicity, hypodermis echogenicity, and deep tissue layer vascularity (P = 0.0014, P = 0.0023, and P = 0.0374, respectively). No significant differences were found for tissue layer thickness or TTS. CONCLUSION: The U-DA may be a useful tool in the identification of localized scleroderma activity. Further study is needed to prospectively evaluate the validity, reliability, and sensitivity of this potential monitoring tool.

Ultrasonography is a sensitive tool for monitoring localized scleroderma.

OBJECTIVE: To examine the usefulness of ultrasonography (USG) for monitoring paediatric localized scleroderma (LS). METHODS: A retrospective chart review of six paediatric patients who had USG of their LS. RESULTS: USG detected several abnormalities in active lesions including increased blood flow, increased echogenicity and loss of subcutaneous fat. USG findings corresponded with clinical assessment, and documented regeneration of subcutaneous fat and reduction in lesion size during treatment. In one patient, USG was more sensitive than magnetic resonance evaluation. CONCLUSION: USG was found to be a sensitive tool for assessing the activity and extent of LS lesions in paediatric patients. Further studies are needed to assess its general applicability for monitoring these patients.

Chlorhexidine gluconate (CHG) activity against clinical isolates of vancomycin-resistant Enterococcus faecium (VREF) and the effects of moisturizing agents on CHG residue accumulation on the skin.

The effectiveness of skin decontamination by chlorhexidine gluconate (CHG) in the presence of commonly-used skin moisturizing lotions was evaluated using vancomycin-resistant Enterococcus faecium (VREF) as a representative nosocomial pathogen. Anti-bacterial efficacy was determined in vitro using pigskin preparations inoculated with five VREF clinical isolates to evaluate Calgon Vestal 2 and 4% (by weight) CHG solutions in comparison with Hibiclens Antiseptic Antimicrobial Cleaner (4% CHG solution). Control inocula were determined for each experiment from recovery of VREF harvested directly from the surface of each control piece of skin. These CHG formulations were evaluated in the presence and absence of Calgon Vestal 'Lotion Soft Skin Conditioner' (LSSC) to determine potential interactions of CHG with LSSC, and also with ¿Vaseline Intensive Care' lotion as a CHG-deactivating agent. The 2% Calgon Vestal CHG alone reduced VREF 10(2)-10(3)-fold, as well as 10(3)-10(4)-fold when LSSC was present, and was as efficacious as either 4% CHG solution when these were tested in the presence of LSSC. Four percent Calgon Vestal CHG produced reductions of 10(3)-10(5)-fold with or without LSSC present. Conversely, ¿Hibiclens' showed similar reductions in the presence of LSSC to that for the Calgon Vestal 4% CHG, but only a 10(1)-10(3)-fold reduction without LSSC. ¿Vaseline Intensive Care' lotion completely inactivated the VREF-killing effects for all of the CHG formulations tested, while LSSC and ¿Vaseline Intensive Care' lotion both showed minimal activity alone against these VREF isolates. These results indicate that the Calgon Vestal 2% CHG solution is as effective against VREF, even in the presence of LSSC, as either the 4% Calgon Vestal or Hibiclens 4% CHG formulations; the use of this lower concentration of CHG may be associated with less irritation, particularly with concomitant use of LSSC.

Rapid oral desensitization to trimethoprim-sulfamethoxazole in infants and children.

BACKGROUND: Although trimethoprim-sulfamethoxazole is the preferred chemoprophylaxis against Pneumocystis carinii pneumonia, there are frequent IgE-mediated reactions among children infected with the human immunodeficiency virus (HIV). Oral desensitization allows more patients to receive chemoprophylaxis, but it has been studied in only a limited number of children. METHODS: We desensitized five children infected with the HIV using a rapid, 4-h oral protocol. RESULTS: Three children (including two infants) successfully completed desensitization and started maintenance therapy, but the other two experienced reactions that precluded further administration of trimethoprim-sulfamethoxazole. CONCLUSIONS: We conclude that a rapid, oral trimethoprim-sulfamethoxazole desensitization protocol is safe and, in some instances, effective among HIV-infected children and infants with a history of non-life-threatening, IgE-mediated reactions to trimethoprim-sulfamethoxazole.

Immunocomplexes stimulate different signalling events to chemoattractants in the neutrophil and regulate L-selectin and beta 2-integrin expression differently.

Neutrophils express receptors for numerous phlogistons which, when occupied, trigger distinct signal-transduction pathways. Previous studies have shown that stimulation of neutrophils with chemoattractants induces shedding of the adhesive molecule L-selectin and increased expression of the beta 2-integrin CD11b/CD18. We determined the effect of ligation of classic, G-protein-linked chemoattractant receptors [C5a, interleukin-8 (IL-8), formylmethionyl-leucylphenylalanine (FMLP) and substance P], receptors for the Fc portion of IgG (Fc gamma receptors) and receptors for transforming growth factor beta (TGF beta) on expression of adhesive molecules by neutrophils and the stimulus-transduction mechanisms thought to mediate these changes. We were surprised to observe that occupancy of Fc gamma receptors by immunocomplexes (BSA-anti-BSA) stimulated increased expression by neutrophils of CD11b/CD18 at concentrations which did not affect L-selectin expression (EC50 9 micrograms/ml versus 350 micrograms/ml respectively, P < 0.00001, n = 5). In contrast, similar to previous studies, recombinant C5a, recombinant IL-8 and FMLP all stimulated increased expression of CD11b/CD18 (170-260% of basal, P < 0.001, n = 5) and shedding of L-selectin (56-75% reduction from basal, P < 0.001, n = 5) at similar concentrations and with similar potencies (EC50 = 2, 5, and 3 nM respectively). In contrast, neither TGF beta 1 nor, surprisingly, substance P affected expression of CD11b/CD18 or L-selectin. The regulation of expression of CD11b/CD18 or L-selectin in response to FMLP or immunocomplexes was unaffected by cytochalasin B (5 micrograms/ml) or the tyrosine kinase inhibitor tyrphostin-25 (25 microM). Although occupancy of both chemoattractant (FMLP) and Fc gamma receptors stimulated increments in the second messenger diacylglycerol, disruption of actin microfilaments by cytochalasin B enhanced diacylglycerol generation in response to FMLP but not in response to ligation of Fc gamma receptors. Moreover, both FMLP and immune aggregates provoked fluxes of intracellular Ca2+ concentration which differed with respect to both magnitude and kinetics and did not correlate well with regulation of adhesive-molecule expression. As upregulation of CD11b/CD18 is tightly linked to exocytosis of specific granules, these results suggest that shedding of L-selectin by activated neutrophils is not linked to exocytosis. These studies provide further evidence that receptors for chemoattractants and immunocomplexes on the neutrophil are linked to multiple signalling pathways.

Chemoattraction of neutrophils by substance P and transforming growth factor-beta 1 is inadequately explained by current models of lipid remodeling.

"Classical" chemoattractants, such as FMLP, C5a, or leukotriene B4, not only elicit directed motility but also activate neutrophils (degranulation, release of active oxygen species). Signal transduction after ligation of receptors for these classical chemoattractants is mediated by pertussis toxin (PT)-sensitive, heterotrimeric G proteins and the early production of lipid messengers via phospholipases. In contrast, we have previously shown that substance P (SP) and transforming growth factor-beta 1 (TGF-beta 1) are "pure" chemoattractants in that they elicit chemotaxis without activating neutrophils. Paradoxically, pure chemoattractants also activate G proteins (plasmalemmal GTPase activity) without eliciting increments in cytosolic calcium ([Ca]i) and thus inositol trisphosphate. We therefore determined lipid remodeling and signal transduction in response to pure chemoattractants. Increments in plasmalemmal GTPase activated by SP (0.1 microM) and TGF-beta 1 (40 fM), like that after FMLP, were PT-sensitive (SP = 6.6 +/- 2 pm/mg/min vs SP + PT = 1.1 +/- 0.9 over basal activity; TGF-beta 1 = 4.3 +/- 1.6 vs TGF-beta 1 + PT = 2.3 +/- 0.9). In parallel, treatment of PMN with PT (1 microgram/ml, 30 min) inhibited chemotaxis (under agarose) after FMLP (2175 +/- 176 (SEM) microns vs 726 +/- 267) and SP (411 +/- 99 microns vs 103 +/- 62 microns) and TGF-beta 1 (40 fM, 375 +/- 53 microns vs 83 +/- 47). However, G proteins coupled to receptors for SP and TGF-beta 1, unlike FMLP, did not appear to be linked to phospholipases in that neither increments in diacylglycerol were detected after receptor ligation (FMLP = 152 +/- 22% resting levels; SP = 101 +/- 5%; TGF-beta 1 = 105 +/- 4%) nor was alkylacylglycerol increased by exposure to SP or TGF-beta 1 (SP = 92 +/- 4%; TGF-beta 1 = 101 +/- 8%; FMLP = 226 +/- 40%). Moreover, polymorphonuclear leukocytes failed to generate phosphatidates (PA) of either species after SP (DA-PA = 79 +/- 9% resting at 60 s; EA-PA = 103 +/- 4%) or TGF-beta 1 (DA-PA = 101 +/- 5%; EA-PA = 98 +/- 9%) in contrast to FMLP (DA-PA = 155 +/- 22%; EA-PA = 149 +/- 16%). The data clearly contravene the current dogma that all chemoattractants use inositol trisphosphate and diglycerides as intracellular signals and suggest the presence of a unique subset of PT-sensitive G proteins, not coupled to "classical" phospholipases, transduce chemoattraction.

Occupancy of G alpha s-linked receptors uncouples chemoattractant receptors from their stimulus-transduction mechanisms in the neutrophil.

Adenosine and adrenergic agonists modulate neutrophil function by ligating their specific receptors (adenosine A2 and beta-adrenergic) on the neutrophil. When occupied, adenosine A2 and beta-adrenergic receptors stimulate, presumably via G alpha s, an increase in intracellular 3', 5' cyclic adenosine monophosphate (cAMP). cAMP affects cellular functions, in part, via protein kinase-mediated phosphorylation. Therefore, we determined whether inhibition of protein kinase A activity by KT5720 (10 mumol/L) reversed the inhibition of FMLP-stimulated O2- generation by 5'N-ethylcarboxamidoadenosine (NECA), the most potent adenosine A2 agonist, and by isoproterenol a potent beta-adrenergic agonist. KT5720 did not affect O2- generation stimulated by FMLP (125% +/- 13% of control, n = 5). However, KT5720 completely reversed inhibition of O2- generation by dibutyryl cAMP (DbcAMP, 1 mmol/L, from 26% +/- 5% to 84% +/- 25% of control, n = 5, P less than .004), but not by NECA (1 mumol/L, 26% +/- 5% v 33% +/- 7% of control, n = 5) or isoproterenol (10 mumol/L, 20% +/- 8% to 38% +/- 6% of control, n = 5). Nearly identical results were obtained using the less specific protein kinase inhibitor H-7. To determine whether occupancy of adenosine A2 or beta-adrenergic receptors inhibits neutrophil (PMN) activation by uncoupling chemoattractant receptors from G proteins, we determined the effect of NECA and isoproterenol on guanosine triphosphatase (GTPase) activity, a parameter that reflects G protein "activation," of plasma membranes derived from human PMNs. Control GTPase activity was 138.9 pmol/mg protein/min; NECA (1 nmol/L to 1 mumol/L) and isoproterenol (10 nmol/L to 10 mumol/L) alone did not significantly affect GTPase activity. FMLP (0.1 mumol/L) increased GTPase activity by 31.9 +/- .9 pmol/mg/min, an increment that was markedly inhibited to approximately 50% of control by NECA (IC50 = 3 nmol/L, P less than .001, n = 5) and isoproterenol (IC50 = 30 nmol/L, P less than .001, n = 5). Neither cAMP nor dibutyryl cAMP (10 mumol/L and 1 mmol/L) affected resting or stimulated GTPase activity. In addition, neither adenosine nor DbcAMP affected protein phosphorylation in resting or stimulated neutrophils. Our studies are consistent with the hypothesis that ligation of G alpha s-linked receptors uncouples chemoattractant receptors from their signal-transduction mechanisms rather than inhibiting neutrophil function via cAMP-mediated effects.

Neuropeptides and inflammation. A somatostatin analog as a selective antagonist of neutrophil activation by substance P.

OBJECTIVE: Substance P and somatostatin are neuropeptides found in peripheral sensory nerves. In vitro, these have opposing effects on inflammatory cells. We compared the effects of these peptides on the activation of neutrophils. METHODS: Neutrophils were isolated from healthy volunteers, and chemotaxis, superoxide anion generation, aggregation, and changes in cytosolic calcium and GTPase activity were measured in the presence of substance P, somatostatin, and the chemoattractant FMLP. RESULTS: Substance P was an effective chemoattractant, 20% as potent as FMLP at equimolar concentrations. Substance P also stimulated GTPase activity in neutrophil plasma membranes. Somatostatin did not activate neutrophils; however, it effectively inhibited neutrophil chemotaxis and GTPase activity provoked by substance P, but not by FMLP. CONCLUSIONS: These studies demonstrate that substance P can effectively stimulate chemotaxis, possibly via effects on a GTP-binding protein distinct from that triggered by FMLP, and that somatostatin is a selective antagonist of substance P. The biochemical specificities of these peptides on cells may modulate neurogenic inflammation at the local level.

Stimulus-response uncoupling in the neutrophil. Adenosine A2-receptor occupancy inhibits the sustained, but not the early, events of stimulus transduction in human neutrophils by a mechanism independent of actin-filament formation.

Generation of superoxide anion (O2-) in response to occupancy of neutrophil chemoattractant receptors requires both early events ('triggering') and sustained signals ('activation'). We have previously demonstrated that occupancy of adenosine A2 receptors inhibits O2- generation by neutrophils. In parallel, adenosine-receptor occupancy promotes association of bound N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors with the cytoskeleton, a process associated with termination of neutrophil activation (stimulus-response uncoupling). We undertook this study to determine whether inhibition of neutrophil function by adenosine-receptor occupancy requires intact actin filaments and to examine the effect of adenosine-receptor occupancy on the stimulated generation of intracellular signals involved in neutrophil triggering and activation. Occupancy of adenosine A2 receptors by 5'-N-ethylcarboxamidoadenosine (NECA, 1 microM) significantly increased (130 +/- 1% of control, P less than 0.001, n = 3) association of [3H]fMLP with cytoskeletal preparations. Cytochalasin B (5 micrograms/ml), an agent which disrupts actin filaments, completely blocked association of [3H]fMLP with cytoskeletal preparations, as previously reported. However, NECA markedly increased association of [3H]fMLP with the cytoskeleton even in the presence of cytochalasin B (P less than 0.0002). Moreover, NECA did not significantly affect either the early (30s) or the late (5 min) formation of actin filaments after stimulation by chemoattractant (fMLP, 0.1-100 nM). Cytochalasin B markedly inhibited actin-filament formation by stimulated neutrophils, and NECA did not reverse the effect of cytochalasin B on actin-filament formation. Adenosine-receptor occupancy did not affect the rapid peak in diacylglycerol generation (less than or equal to 15 s) from either [3H]arachidonate- or [14C]glycerol-labelled phospholipid pools. However, as would be predicted if occupancy of the adenosine receptor was a signal for early termination of cell activation, NECA (1 microM) markedly diminished the slow sustained generation of diacylglycerol. These results suggest that adenosine-A2-receptor occupancy does not affect triggering of the neutrophil, but that occupancy of adenosine receptors is an early signal for the termination of neutrophil activation, i.e. the 'premature' finish of signal transduction. Moreover, these data indicate that at least two pathways are available for increasing the association of ligated chemoattractant receptors with the cytoskeleton of neutrophils: F-actin-dependent and -independent.

Effects of protein I of Neisseria gonorrhoeae on neutrophil activation: generation of diacylglycerol from phosphatidylcholine via a specific phospholipase C is associated with exocytosis.

Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways.

Transforming growth factor beta 1 (TGF-beta 1), a homodimeric polypeptide (Mr 25,000), derives from inflammatory cells and acts as a chemoattractant for monocytes and fibroblasts. We report here that TGF-beta 1 is also the most potent chemoattractant yet described for human peripheral blood neutrophils. Recombinant TGF-beta 1 elicited dose-dependent directed migration of neutrophils under agarose that was inhibited in the presence of a neutralizing antibody to TGF-beta 1. Maximal chemotaxis was evoked by TGF-beta 1 at femtomolar concentrations, whereas conventional chemoattractants act at nanomolar concentrations: on a molar basis, TGF-beta 1 was 150,000 times more potent than fMet-Leu-Phe. In contrast, TGF-beta 1 provoked neither exocytosis nor the production of superoxide by neutrophils. We further analyzed the mechanism by which TGF-beta 1 elicits chemotaxis (GTPase activity, [Ca2+], and actin polymerization). In contrast to the conventional chemoattractant fMet-Leu-Phe, TGF-beta neither activated classic heterotrimeric guanine nucleotide-binding proteins nor provoked global mobilization of intracellular Ca2+. Chemoattraction by both fMet-Leu-Phe and TGF-beta 1 was inhibited by cycloheximide and actinomycin D. Moreover, chemotaxis in response to TGF-beta 1 was associated with the polymerization of actin. The selectivity and potency of TGF-beta 1 as a chemoattractant suggest that it elicits directed cell migration by means of a pathway that depends not on classic intracellular signals but on protein synthesis.

Differences in signal transduction between Fc gamma receptors (Fc gamma RII, Fc gamma RIII) and FMLP receptors in neutrophils. Effects of colchicine on pertussis toxin sensitivity and diacylglycerol formation.

Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis.

Low molecular weight GTP-binding proteins in human neutrophil granule membranes.

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 blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase.

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.

Activation of the human neutrophil by calcium-mobilizing ligands. II. Correlation of calcium, diacyl glycerol, and phosphatidic acid generation with superoxide anion generation.

Calcium and protein kinase C (Ca2+/phospholipid-dependent enzyme) have been proposed to act as signals in triggering superoxide anion (O2-) generation by neutrophils. We have probed the adequacy and necessity of calcium and diacylglycerol (DG), activators of protein kinase C, in eliciting O2- generation and degranulation. Activation of neutrophils by the ligand 10(-7) M fMet-Leu-Phe triggered elevation of cytosolic calcium (fura-2) and a rapid, biphasic increase in labeled DG in [14C]glycerol and [3H]arachidonate prelabeled cells. Buffering of the fMet-Leu-Phe-induced elevation of cytosolic calcium with MAPTAM (a cell permeant EGTA analogue) inhibited O2- generation by 90% and degranulation by 50%, concordant with a role of calcium in signaling. However, buffering the increase in calcium also decreased DG. Since phosphatidylinositol 4,5-bisphosphate breakdown in response to fMet-Leu-Phe was not inhibited and phosphatidic acid levels were enhanced in MAPTAM pretreated cells, the removal of calcium may enhance further DG metabolism. Thus, a requirement for calcium could not be differentiated from a requirement for DG, and the profound inhibition of O2- generation in the presence of MAPTAM may reflect removal of DG. Four stimuli, fMet-Leu-Phe, 10(-7) M leukotriene B4, 100 micrograms/ml concanavalin A, and 200 nM ionomycin elevated cytosolic calcium and triggered release of specific granules, but only fMet-Leu-Phe and concanavalin A triggered substantial O2- generation. Nevertheless, all four stimuli significantly increased labeled DG. Therefore, elevated DG and elevated calcium may be necessary but do not appear adequate to elicit O2- generation. Only fMet-Leu-Phe and concanavalin A triggered generation of phosphatidic acid (PA) together with DG. Correlation of O2- generation with PA may reflect a requirement for PA per se or for a specific pool of DG that can be further metabolized to PA.

Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish "triggering" from "activation" of human neutrophils.

Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O2-) which are potent mediators of inflammation. Various agents elicit different responses; N-formylmethionylleucylphenylalanine (fMLP) (0.1 microM) provokes brisk generation of superoxide anion; leukotriene B4 (LTB4, 0.1 microM) is a poor stimulus. In contrast, phorbol myristate acetate (PMA, 1.6 microM) acting directly via protein kinase C is a potent stimulus for O2-. We compared the kinetics of appearance of various "second messengers" with the capacity of these ligands to elicit O2- generation. Kinetic analysis showed a two-phase response to membrane ligands; both an "early" (less than or equal to 15 s) and a "late" (greater than 15 s) increase in [3H]- and [14C]diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB4 elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Cytochalasin B increased the late phase of DG labeling elicited by all agonists. Moreover, comparison of increases in [3H]DG versus those of [14C]DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Early increments of DG were also accompanied by addition of plasma membrane (ultrastructural morphometry); the ratio of surface perimeter to area increased rapidly (10 s) and persisted (60 s) in response to fMLP. Increments were more gradual in response to PMA. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 Mr protein was phosphorylated with kinetics consistent with the production of O2- and DG in response to fMLP (early and late) and PMA (late). In contrast, LTB4 provoked only early phosphorylation of this protein. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only "triggering" (the rapid generation of a signal) but also "activation" (the maintenance of a signal) to sustain responses.

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation.

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.