Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing.

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)

Mycobacterium tuberculosis induces CCL18 expression in human macrophages.

The interaction of Mycobacterium tuberculosis (MTB) with the immune system is mediated by cytokine and chemokine responses of macrophages and/or dendritic cells. Chemokine (C-C motif) ligand 18 (CCL18) and interleukin (IL)-10 are major factors secreted by phagocytes, postulated to recruit naïve T lymphocytes and inhibit pro-inflammatory cells. Our study investigated the role of CCL18 and IL-10 in an in vitro model of infection by MTB in human macrophages. CD14(+) monocytes, obtained from the peripheral blood of eight healthy donors, differentiated in monocyte-derived macrophages (MDM) with monocyte-colony stimulating factor (100 ng/ml) for 6 days, were stimulated in vitro with lipopolysaccharide (LPS) (1 microg/ml) and with heat killed MTB Hv37Ra (multiplicity of infection 1:5) for 24 h. Alveolar macrophages from five healthy donors were infected with MTB Hv37RA. CCL18 protein and mRNA were detected by enzyme-linked immunosorbent assay (ELISA) and real-time PCR, IL-10 levels by ELISA. Stimulation of MDM with LPS or MTB led to a significant increase in CCL18 protein (control 2.67 +/- 0.46 ng/ml, LPS 4.05 +/- 0.56 ng/ml, with MTB 6.70 +/- 1.59 ng/ml, n = 5, P < 0.05) and specific mRNA levels (control 0.09 +/- 0.01, LPS 0.24 +/- 0.11, with MTB 0.34 +/- 0.08 CCL18/Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), n = 3, P < 0.05). A significant increase of the production of CCL18 was observed in infected alveolar macrophages. IL-10 levels increased from 38.52 +/- 26.38 pg/ml in control cells to 1129.32 +/- 235.00 and 974.25 +/- 164.46 pg/ml in LPS and MTB treated cells, respectively (P < 0.05). Up-regulation of CCL18 and IL-10 in macrophages by MTB may be involved in the recruitment of naïve T cells in association with local suppressive immunity against intracellular pathogens. This could represent a mechanism of tolerance during the early phases of infection.

Enhanced interleukin-8 release and gene expression in macrophages after exposure to Mycobacterium tuberculosis and its components.

Mycobacterium tuberculosis infection is accompanied by acute and chronic inflammatory infiltrates associated with necrotizing granulomas in lung tissue. The cellular infiltrate is characterized by inflammatory cells which include neutrophils, lymphocytes, and macrophages. In animal and in vitro models of mycobacterial infection, cytokines including tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin-1 beta (IL-1 beta) participate in granulomatous inflammation. We hypothesized that interleukin-3, a potent chemoattractant for neutrophils and lymphocytes, could be released by activated alveolar macrophages after exposure to M. tuberculosis or its components and contribute to granulomatous lung inflammation. A quantitative immunoassay revealed that IL-8 protein release was significantly elevated in supernatants of macrophages and in lavage fluid obtained from patients with pulmonary tuberculosis compared to normal controls. In addition, Northern blots demonstrated striking up-regulation of IL-8 mRNA in macrophages from these patients. M. tuberculosis and its cell wall components lipoarabinomannan (LAM), lipomannan (LM), and phosphoinositolmannoside (PIM) stimulated IL-8 protein release and mRNA expression in vitro from alveolar macrophages, but deacylated LAM did not. Neutralizing antibodies to TNF-alpha and/or IL-1-alpha and beta blocked 83% of the stimulation. IL-8 synthesis and release is an early response of macrophages after phagocytosis of M. tuberculosis. Its production serves to attract both acute and chronic inflammatory cells of active infection and thus participates in the process of containment of the pathogen.

Colchicine alters the quantitative and qualitative display of selectins on endothelial cells and neutrophils.

Since colchicine-sensitive microtubules regulate the expression and topography of surface glycoproteins on a variety of cells, we sought evidence that colchicine interferes with neutrophil-endothelial interactions by altering the number and/or distribution of selectins on endothelial cells and neutrophils. Extremely low, prophylactic, concentrations of colchicine (IC50 = 3 nM) eliminated the E-selectin-mediated increment in endothelial adhesiveness for neutrophils in response to IL-1 (P < 0.001) or TNF alpha (P < 0.001) by changing the distribution, but not the number, of E-selectin molecules on the surface of the endothelial cells. Colchicine inhibited stimulated endothelial adhesiveness via its effects on microtubules since vinblastine, an agent which perturbs microtubule function by other mechanisms, diminished adhesiveness whereas the photoinactivated colchicine derivative gamma-lumicolchicine was inactive. Colchicine had no effect on cell viability. At higher, therapeutic, concentrations colchicine (IC50 = 300 nM, P < 0.001) also diminished the expression of L-selectin on the surface of neutrophils (but not lymphocytes) without affecting expression of the beta 2-integrin CD11b/CD18. In confirmation, L-selectin expression was strikingly reduced (relative to CD11b/CD18 expression) on neutrophils from two individuals who had ingested therapeutic doses of colchicine. These results suggest that colchicine may exert its prophylactic effects on cytokine-provoked inflammation by diminishing the qualitative expression of E-selectin on endothelium, and its therapeutic effects by diminishing the quantitative expression of L-selectin on neutrophils.

Prevalence of workplace exacerbation of asthma symptoms in an urban working population of asthmatics.

OBJECTIVES: We used an interviewer-administered questionnaire to investigate workplace exacerbation of asthma symptoms (WEAS) among low-income, minority, working asthmatics admitted Bellevue Hospital Center in New York City from 2001 to 2002. We hypothesized that a high prevalence of WEAS would be found in this population among all jobs held and a subset of individual occupational classifications. MEASUREMENTS AND MAIN RESULTS: Of 301 subjects, 51% reported WEAS in their current or most recent job; 71% reported WEAS in any job. Prevalences (95% confidence intervals) of WEAS in common job classifications were 61% (49-73%) in janitorial jobs, 50% (33-67%) in garment and textile manufacturing jobs, and 38% (23-55%) in construction jobs. CONCLUSION: WEAS is prevalent in this urban minority population.

Non-steroidal anti-inflammatory drugs: effects on a GTP binding protein within the neutrophil plasma membrane.

Sodium salicylate and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via unknown mechanisms. To examine their site of action in the neutrophil we have studied discrete events within the plasma membrane which depend upon the normal function of a GTP binding protein (G protein). We demonstrated that sodium salicylate and piroxicam inhibit neutrophil activation in response to stimuli which require signal transduction via a G protein (e.g. formyl-methionine-leucine-phenylalanine) but have no effect on stimuli which do not (e.g. phorbol myristate acetate, ionomycin). NSAIDs blocked the ADP-ribosylation of the pertussis toxin substrate in human neutrophils. This effect was associated with the capacity of NSAIDs to block pertussis toxin-dependent inhibition of neutrophil functions. Finally, NSAIDs inhibited the binding of GTP gamma S, a stable analog of GTP, to purified neutrophil membrane preparations. The data indicate that salicylate and other NSAIDs interact with a G protein in the neutrophil plasmalemma and thereby uncouple post-receptor signaling events.

Immunohistochemical localization of transforming growth factor beta isoforms in asbestos-related diseases.

Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases.

Esmolol and percutaneous cardiopulmonary bypass enhance myocardial salvage during ischemia in a dog model.

Despite recent advances in techniques of reperfusion for acute myocardial ischemia, myocardial salvage remains suboptimal. Beta-blockers have been shown to limit infarct size during acute ischemia, but their negative inotropic properties have limited their use. Cardiopulmonary bypass is an attractive technique for cardiac resuscitation because it can stabilize a hemodynamically compromised patient and potentially reduce myocardial oxygen consumption. In an attempt to maximize myocardial salvage in the setting of acute ischemia, the combination of esmolol, an ultrashort-acting beta-blocker, with percutaneous cardiopulmonary bypass was evaluated. Four groups of instrumented dogs underwent 2 hours of myocardial ischemia induced by occlusion of the proximal left anterior descending coronary artery, followed by 1 hour of reperfusion. Throughout the period of ischemia and reperfusion, esmolol plus percutaneous cardiopulmonary bypass was compared with esmolol alone, percutaneous cardiopulmonary bypass alone, and control conditions. After the reperfusion period, the extent of infarction of the left ventricle at risk was determined. Four animals had intractable arrhythmias: one in the esmolol plus bypass group, one in the esmolol group, and two in the control group. The extent of infarction of the left ventricle at risk was significantly reduced in the esmolol plus bypass group (30%) compared with bypass alone (52%), with esmolol alone (54%), and with the control groups (59%; p < 0.05). We conclude that in this experimental model the combination of esmolol with bypass improves myocardial salvage after ischemia and reperfusion.

Reduction in pulmonary microvascular pressure following cardiopulmonary bypass: beneficial effects of dobutamine.

Pulmonary microvascular pressures (PMVP) have important diagnostic and therapeutic implications when utilized to monitor pulmonary dysfunction after cardiopulmonary bypass. Elevations in PMVP may lead to interstitial pulmonary edema and right ventricular failure. This study evaluated the influence of Dobutamine on PMVP in a trial of 80 consecutive patients undergoing isolated coronary artery bypass grafting (CABG). Forty patients were randomized to the Dobutamine study group and received 5 micrograms/kg/min of Dobutamine for 24 hours, starting at the completion of bypass. In the control group, patients received postoperative inotropic support as indicated (dopamine [n = 10] or amrinone [n = 6]) by the clinical situation. PMVP values were computed based on continuous hemodynamic monitoring at 6, 12, 18 and 24 hours. Preoperative demographic descriptors and operative variables were comparable between the two groups. Postoperative fluid requirements and nonpulmonary complications were also similar between groups. Upon completion of cardiopulmonary bypass, PMVP (mean +/- SD) were PMVP decreased over time in the Dobutamine group, while it did not change in the control group. Clinically mean time to extubation was reduced from 18 to 12 hours (p < 0.06) in the Dobutamine group. We conclude that in patients undergoing cardiopulmonary bypass, the postoperative administration of Dobutamine significantly reduces the PMVP. This may reduce pulmonary interstitial edema and pulmonary complications. Upon completion of cardiopulmonary bypass, PMVP (mean +/- SD) were measured at 6 hours, 12 hours, 18 hours and 24 hours. The control group measured 25 +/- 5 mmHg, 26 +/- 2 mmHg, 27 +/- 3 mmHg and 28 +/- 3 mmHg. The Dobutamine group measured 25 +/- 6 mmHg, 24 +/- 3 mmHg, 22 +/- 2 mmHg and 18 +/- 5 mmHg. PMVP decreased over time in the Dobutamine group (p < 0.001), while it did not change in the control group. Clinically mean time to extubation was reduced from 18 to 12 hours (p < 0.06) in the Dobutamine group. We conclude that in patients undergoing cardiopulmonary bypass, the post-operative administration of Dobutamine significantly reduced PMVP. This may reduce pulmonary interstitial edema and pulmonary complications post cardiopulmonary bypass.

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.

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.

Mast cell degranulating (MCD) peptide analogs with reduced ring structure.

Mast cell degranulating (MCD) peptide, a component of bee venom, is a 22 amino acid peptide with two disulfide bridges. In this first structure-activity study of MCD peptide, three analogs were synthesized and tested: two analogs shortened by omitting sequences 6-10 and 8-13, respectively, and one analog lacking the disulfide bridge between cysteine residues 5 and 19. These analogs were synthesized by solid-phase methods and were compared to MCD peptide in two assays for inflammation: histamine release from mast cells and superoxide anion release from neutrophils. All three analogs produced histamine release, although with only about one fifth of the activity of MCD peptide. Superoxide anion-releasing activity, however, did not parallel histamine release. MCD peptide did not release superoxide anion, while the 6-10 and 8-13 deletion analogs were strong and weak stimulants, respectively, of this anion. CD spectra showed that the secondary structures of the three analogs were very similar to that of MCD peptide, so that a change in secondary structure cannot completely explain the changes in releasing activities. Charge differences between the two deletion analogs and MCD peptide may explain some of the differences in activity. This is the first demonstration that the various activities of MCD peptide can be separated, and provides a lead through which the purported antiinflammatory activity of MCD peptide may possibly be explored in the future.

Cigarette smoking and ozone-associated emergency department use for asthma by adults in New York City.

The association between ambient ozone (O3) and hospital use for asthma in children and adults is well documented. The question remains of whether there are susceptible subpopulations of asthmatic individuals who are particularly vulnerable to high O3 levels. Because tobacco use was prevalent in our cohort of inner-city adult asthmatic individuals (n = 1,216) in New York City (NYC), we investigated whether cigarette smoking was an effect modifier for asthma morbidity. We examined the relationship between personal tobacco use and O3-associated emergency department (ED) use for asthma in public hospitals in NYC. Three subpopulations were defined: never smokers (0 pack-yr), heavy smokers (>/= 13 pack-yr) and light smokers (< 13 pack-yr). Time-series regression analysis of ED use for asthma and daily O3 levels was done while controlling for temperature, seasonal/long-term trends, and day-of-week effects. Heavy smokers displayed an increased relative risk (RR) of ED visits for asthma in response to increases in 2-d lagged O3 levels (RR per 50 ppb O3 = 1.72; 95% confidence interval: 1.13 to 2.62). Logistic regression analysis confirmed that heavy cigarette use was a predictor of ED use for asthma following days with high O3 levels. Although adverse health effects of ambient O3 have also been documented in asthma populations not using cigarettes (e.g., children), our results suggest that in adult asthmatic individuals, heavy personal tobacco use may be an effect modifier for O3-associated morbidity.

Early inhibition of mycobacterial growth by human alveolar macrophages is not due to nitric oxide.

Phagocytic cells provide the first line of defense against mycobacteria. We examined the relative mycobacteriostatic contributions of normal human alveolar macrophages (HAM), peripheral blood monocytes (PBM), and polymorphonuclear leukocytes (PMN) in the early time period after infection with mycobacteria (48 h). Cells were infected with Mycobacterium bovis (BCG) or M. tuberculosis H37Ra and their ability to inhibit growth was determined by mycobacterial incorporation of [3H]uracil. HAM inhibited the growth of both mycobacteria (44.2 +/- 7.9 and 37.6 +/- 10.5% inhibition, respectively). Two populations of HAM donors were subsequently defined: inhibitors and noninhibitors. The ability to inhibit growth of H37Ra correlated with that of BCG. In contrast to HAM, PBM and PMN did not inhibit mycobacterial growth. Because nitric oxide (NO) has been proposed to mediate growth inhibition in murine models, we examined whether NO was responsible for the early growth inhibition of mycobacteria by HAM. As expected, in murine peritoneal macrophages (MPM) IFN-gamma (2,500 U/ml) enhanced growth inhibition of BCG; the effect was abolished by the nitric oxide synthase (NOS) inhibitor NMMA. In contrast, IFN-gamma failed to enhance growth inhibition by HAM or PBM and NMMA had no effect. MPM expressed inducible nitric oxide synthase (NOS2) mRNA in response to LPS and IFN-gamma and produced NO. Neither NOS2 mRNA nor NO could be detected in HAM stimulated with LPS and IFN-gamma or mycobacteria. These data demonstrate that HAM, but not PBM or PMN, have NO-independent mycobacteriostatic activity in the early time period after infection with mycobacteria.

Separation of younger red cells with improved survival in vivo: an approach to chronic transfusion therapy.

Transfusion of donor blood containing predominantly younger red cells with prolonged survival in vivo could significantly reduce the iron overload of patients requiring chronic transfusion. Age-dependent separation of red cells can be obtained by buoyant density centrifugation on isotonic solutions of arabino-galactane. By this technique, rabbit red cells were separated on a single layer of arabino-galactane and the appropriate fraction, after being labeled with (51)Cr, was reinfused into the donor. The survival in vivo was calculated by a mathematical model which corrects for both (51)Cr elution and random loss. There was a significant difference in survival in vivo between the light young red cells and the heavy old red cells. The potential survival in vivo of the 50% lightest red cells was 56 days, compared to 28 days for the heaviest red cells. Arabino-galactane appeared to be devoid of acute toxicity and of strong antigenicity and it did not appear to adhere to the red cell stroma. These data extrapolated to humans indicate that it may be feasible and advantageous to use red cells fractionated by this technique for transfusion. The 50% lightest human red cells can be expected to have a mean survival of 88 days, compared with 60 days for unfractionated blood. Transfusion of young red cells could significantly reduce the iron overload for patients requiring chronic transfusion, by avoiding infusion of the oldest red cells, which contribute equally to iron overload yet offer only transient survival in vivo.

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin.

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.

Effect of maternal asthma on performance of parenting tasks and children's school attendance.

We evaluated the effects of maternal asthma on specific parameters of family function including the children's school attendance and mother's performance of basic parenting tasks. A case-controlled study of mothers with asthma (MA; n = 24) with children under the age of 13 and matched mothers without asthma (CM; n = 27) was performed. Children of mothers with asthma had a significantly impaired ability to attend school compared to children of control mothers (odds ratio = 15, 95% CI). Twenty-two percent of MA reported that their asthma caused their children to miss school at least once per month. In addition, 27% of MA reported that their children were regularly late for school because of the mother's asthma. Only 5% of the control mothers reported that their health caused their children to miss school, and none reported lateness. Asthma also impaired the ability of the MA to perform basic parenting tasks such as dressing children and preparing meals for children. These adverse effects of parental asthma on children's school attendance and parenting represent previously unappreciated indirect costs of asthma and may have immediate as well as future consequences.

Regulation of expression of granulocyte-macrophage colony-stimulating factor in human bronchial epithelial cells: roles of protein kinase C and mitogen-activated protein kinases.

GM-CSF has a major role in the immune and inflammatory milieu of the airway. Airway epithelial cells (AEC) are among the first targets of environmental stimuli and local cytokines, in response to which they can produce GM-CSF. The regulation of GM-CSF is only minimally understood in AEC. We hypothesized that GM-CSF expression in AEC would result from activation of protein kinase C (PKC) and subsequent activation of the extracellular signal-regulated kinase (MAPKerk1/2) pathway, so we investigated signal transduction pathways in human primary culture bronchial epithelial cells (HBECs). TNF-alpha, IL-1beta, and PMA induced the release of GM-CSF in HBECs. The robust response to PMA was not detected in SV40 adenovirus-transformed normal human bronchial epithelial cells (BEAS-2B). PMA and TNF-alpha stimulation of GM-CSF required activation of PKC (inhibition by staurosporine and bisindolylmaleimide I). GM-CSF expression was up-regulated by a nonphorbol PKC activator, but not by an inactive PMA analogue. PMA-induced GM-CSF production in HBECs did not require a Ca2+ ionophore and was not inhibited by cyclosporin A. Activation of MAPKerk1/2 via PKC was associated with and was required for GM-CSF production induced by PMA and TNF-alpha. The data demonstrate regulation of GM-CSF in HBECs by PKC pathways converging on the MAPKerk1/2 pathway and further define cell-specific regulation critical for local airway responses.