Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.     

Macrophage-derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of resting T cells

To examine how macrophage-derived nitric oxide (NO) affects T helper (Th) cell activity, T cell clones representing Th1 and Th2 subsets were activated before exposure to stimulated peritoneal macrophages or microglia. Both Th subsets were similarly sensitive to inhibition by NO, indicating that macrophage-derived NO regulates the proliferation of activated Th1 and Th2 cells equally well. Since IFN-gamma production remained intact in NO-treated Th1 cells, we studied whether NO was produced during antigen-specific activation of Th1 cells by unstimulated macrophages. Indeed, T cell proliferation only occurred when a NO synthase inhibitor was included, while IFN-gamma was essential for the induction of NO. These studies demonstrate that macrophages produce NO following antigen presentation to Th1 cells and that macrophage-derived NO inhibits Th1 and Th2 cell proliferation without inhibiting cytokine production.     

Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation.

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323-339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-gamma. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.

Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.     

A schistosome-expressed immunomodulatory glycoconjugate expands peritoneal Gr1(+) macrophages that suppress naive CD4(+) T cell proliferation via an IFN-gamma and nitric oxide-dependent mechanism

Lacto-N-fucopentaose III (LNFPIII) is found in human milk and on the Th2 driving helminth parasite Schistosoma mansoni. This pentasaccharide drives Th2-type responses in vivo and in vitro when conjugated to a carrier. In an attempt to further understand early events in Th1 to Th2 switching, we examined phenotypic and functional changes in peritoneal cell populations in BALB/c and SCID mice following LNFPIII-dextran injection. We found that i.p. injection with LNFPIII-dextran resulted in rapid (<20 h) expansion of the Gr1(+) subpopulation of F4/80(+)/CD11b(+) peritoneal cells, comprising up to 75% of F4/80(+)/CD11b(+) peritoneal cells compared with 18% in uninjected or dextran-injected mice. Functionally, these cells suppressed anti-CD3- and anti-CD28-induced proliferation of naive CD4(+) T cells. LNFPIII-dextran also expanded functional Gr1(+) suppressor macrophages in SCID mice, demonstrating that expansion and function of suppressor cells did not require T cells. Suppression in both BALB/c and SCID mice was NO and IFN-gamma dependent, as addition of inhibitors of inducible NO synthase (N(G)-monomethyl-L-arginine), as well as anti-IFN-gamma Abs, restored the ability of CD4(+) T cells to proliferate in vitro. Depletion of the F4/80(+) subset of Gr1(+) cells eliminated the suppressive activity of peritoneal exudate cells showing that these cells were macrophages. Thus, LNFPIII-dextran rapidly expands the Gr1(+) suppressor macrophage population in the peritoneal cavities of otherwise naive mice. These Gr1(+) cells suppress proliferation of naive CD4(+) T cells in an NO-dependent mechanism, and may play a regulatory role in the switching of Th1- to Th2-type responses.     

Defining the molecular role of gp91phox in the immune manifestation of acute allergic asthma using a preclinical murine model

ABSTRACT: OBJECTIVE: The phenomena manifested during inflammation require interplay between circulating effector cells, local resident cells, soluble mediators and genetic host factors to establish, develop and maintain itself. Of the molecues involed in the initiation and perpetuation of acute allergic inflammation in asthma, the involvement of effector cells in redox reactions for producing O2- (superoxide anion) through the mediation of NADPH oxidase is a critical step. Prior data suggest that reactive oxygen species (ROS) produced by NADPH oxidase homologues in non-phagocytic cells play an important role in the regulation of signal transduction, while macrophages use a membrane-associated NADPH oxidase to generate an array of oxidizing intermediates which inactivate MMPs on or near them. MATERIALS AND METHODS AND TREATMENT: To clarify the role of gp91phox subunit of NADPH oxidase in the development and progression of an acute allergic asthma phenotype, we induced allergen dependent inflammation in a gp91phox-/- single knockout and a gp91phox-/-MMP-12-/- double knockout mouse models. RESULTS: In the knockout mice, both inflammation and airway hyperreactivity were more extensive than in wildtype mice post-OVA. Although OVA-specific IgE in plasma were comparable in wildtype and knockout mice, enhanced inflammatory cell recruitment from circulation and cytokine release in lung and BALf, accompanied by higher airway resistance as well as Penh in response to methacholine, indicate a regulatory role for NADPH oxidase in development of allergic asthma. While T cell mediated functions like Th2 cytokine secretion, and proliferation to OVA were upregulated synchronous with the overall robustness of the asthma phenotype, macrophage upregulation in functions such as proliferation, and mixed lymphocyte reaction indicate a regulatory role for gp91phox and an overall non-involvement or synergistic involvement of MMP12 in the response pathway (comparing data from gp91phox-/- and gp91phox-/-MMP-12-/- mice).     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Superoxide anion (O2-*) production is elevated in sympathetic ganglion neurons and in the vasculature of hypertensive animals; however, it is not known what enzymatic pathway(s) are responsible for O2-* production. To determine the pathway(s) of O2-* production in sympathetic neurons, we examined the presence of mRNA of NADPH oxidase subunits in sympathetic ganglionic neurons and differentiated PC-12 cells. The mRNAs for NADPH oxidase subunits p47phox, p22phox, gp91phox, and NOX1 were present in sympathetic neurons and PC-12 cells, whereas the NOX4 homologue was present in sympathetic neurons but not PC-12 cells. Freshly dissociated celiac ganglion neurons from normal rats and PC-12 cells produced O2-* when treated with the PKC activator PMA; O2-* production increased by 317% and 254%, respectively. The PMA-evoked increases were reduced by pretreatment with the NADPH oxidase inhibitor apocynin. These findings indicate that NADPH oxidase is the primary source of O2-* in sympathetic ganglion neurons. When celiac ganglia from hypertensive rats were incubated with apocynin, O2-* levels were reduced to the same levels as normotensive animals, indicating that NADPH oxidase activity accounted for the elevated O2-* levels in hypertensive animals. To test this latter finding, we compared NADPH oxidase activity in extracts of prevertebral sympathetic ganglia of DOCA-salt hypertensive rats and sham-operated rats. NADPH oxidase activities were 49.9% and 78.6% higher in sympathetic ganglia of DOCA rats compared with normotensive controls when using beta-NADH and beta-NADPH as substrates, respectively. Thus elevated O2-* levels in hypertension may be a result of the increased activity of NADPH oxidase in postganglionic sympathetic neurons.     

Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion

To study the effect of nitric oxide (NO) on the activity of Th subsets, cloned Th1 and Th2 lymphocytes were stimulated in the presence of an NO donor. NO, when present from the start of incubation, inhibited the proliferation of both Th subsets dose-dependently, achieving complete inhibition at a relatively low level. The addition of NO 24 h after the onset of T cell stimulation also resulted in reduced proliferation of both Th subsets, suggesting that NO affects a late process during T cell activation. Stimulation of T cells in the presence of NO did not induce apoptosis at the concentrations that completely inhibited proliferation, although apoptosis became evident at higher NO concentrations. The secretion of several cytokines (i.e., IFN-gamma, IL-4, and IL-5) was slightly upregulated, while IL-2 production was modestly inhibited in the presence of NO. However, exogenous IL-2 did not reverse the NO-induced inhibition of T cell proliferation, nor did additional stimulation with phorbol esters. Finally, expression of IL-2R was modestly decreased in the presence of NO, although TCR expression was not affected. These studies demonstrate that relatively low concentrations of NO induce a strong and specific inhibition of T cell proliferation in both Th subsets, suggesting that local NO production may regulate Th-mediated tissue inflammation.     

Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression

Alveolar macrophages, which generate high levels of reactive oxygen species, especially O(2)(*-), are involved in the recruitment of neutrophils to sites of inflammation and injury in the lung, and the generation of chemotactic proteins triggers this cellular recruitment. In this study, we asked whether O(2)(*-) generation in alveolar macrophages had a role in the expression of chemokines. Specifically, we hypothesized that O(2)(*-) generation is necessary for chemokine expression in alveolar macrophages after TNF-alpha stimulation. We found that alveolar macrophages have high constitutive NADPH oxidase activity that was not increased by TNF-alpha, but TNF-alpha increased the activity of the mitochondrial respiratory chain. In addition, the mitochondrial respiratory chain increased O(2)(*-) generation if the NADPH oxidase was inhibited. O(2)(*-) generation was necessary for macrophage inflammatory protein-2 (MIP-2) gene expression, because inhibition of NADPH oxidase or the mitochondrial respiratory chain or overexpression of Cu,Zn-superoxide dismutase significantly inhibited expression of MIP-2. TNF-alpha activated the ERK MAP kinase, and ERK activity was essential for chemokine gene expression. In addition, overexpression of the MEK1-->ERK pathway significantly increased IL-8 expression, and a small interfering RNA to the NADPH oxidase inhibited ERK- and TNF-alpha-induced chemokine expression. Collectively, these results suggest that in alveolar macrophages, O(2)(*-) generation mediates chemokine expression after TNF-alpha stimulation in an ERK-dependent manner.     

Comparison of 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate as secretagogues for human neutrophils

Cellular responses induced in human neutrophils by the synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), paralleled those induced by phorbol myristate acetate (PMA). Like PMA, OAG caused the preferential release of enzymes from specific granules and promoted superoxide (O2-) generation. The efficacy of OAG was similar to that for PMA, but its potency was lower by four orders of magnitude. First derivative kinetic analysis showed that rates of O2- generation elicited by PMA decayed exponentially in a first order manner; the half life was found to be 21 +/- 6 min. Results obtained in studies carried out with high OAG concentrations were similar except that after 40 min, the rate of decay increased and became complex order. This difference was attributed to the greater susceptibility of OAG to metabolic alteration, and was reflected in the NADPH oxidase activity of granule rich membrane fractions (GRF) of cells stimulated for 90 min with PMA or OAG. It was found that the O2- generating activity of the PMA treated GRF was significantly greater than that for the OAG treated fraction. Current evidence indicates that cellular responses arise from direct activation of protein kinase C by PMA-OAG. The stability of this complex and the bypassing of normal regulatory constraints may account for the relative longevity of the PMA-OAG O2- respiratory burst.     

Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils

Gliotoxin from Aspergillus, bearing a S&bond;S bond in its structure, prevented the onset of O(-)(2) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O(-)(2) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC(50)) of 5.3, and 3.5 microM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC(50) of 0.009 microM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.     

House dust mite Dermatophagoides farinae augments proinflammatory mediator productions and accessory function of alveolar macrophages: implications for allergic sensitization and inflammation

In this study, we examine the effects of Dermatophagoides farinae (Der f), a major source of airborne allergens, on alveolar macrophages (AMs), and we also test its contribution to allergic responses in mice. Der f activated NF-kappaB of AMs and, unlike OVA or LPS stimulation, up-regulated IL-6, TNF-alpha, and NO. In addition, it down-regulated antioxidants, but affected neither the expression nor production of IL-12. Der f-stimulated AMs expressed enhanced levels of costimulatory B7 molecules, supported T cell proliferation, and promoted Th2 cell development. The enhanced accessory function was suppressed by blockade mAbs to B7.2, IL-6, and TNF-alpha and by N-monomethyl-L-arginine, an NO synthase inhibitor, and N-acetylcysteine, a thiol antioxidant, whereas it was augmented by (+/-)-S-nitroso-N-acetylpenicillamine, an NO donor. Arg-Gly-Asp-Ser peptide and neo-glycoproteins galactose-BSA and mannose-BSA inhibited the Der f-induced IL-6 and TNF-alpha productions and enhanced accessory function of AMs. Der f was more potent than OVA for inducing pulmonary eosinophilic inflammation, NO, and serum allergen-specific IgG1 Ab production in mice. AMs from Der f-challenged mice expressed enhanced levels of B7 and augmented T cell proliferation ex vivo. In Der f-challenged mice, respiratory syncytial virus infection (5 x 10(5) pfu; 3 days before Der f instillation) augmented Der f-specific Ab production, whereas dexamethasone (50 mg/kg; 1 h before Der f instillation) diminished the allergic airway inflammation and Ab response. We conclude that AMs are sensitive targets for Der f and that the Der f-induced proinflammatory responses may represent an important mechanism in mediating the development of allergic sensitization and inflammation.     

Age-related alterations in superoxide anion generation in mouse peritoneal macrophages studied by repeated stimulations and heat shock treatment.

The ability of thioglycollate-elicited peritoneal macrophages (PM) from young and senescent mice to generate superoxide anions (O2-) under repeated stimulation or thermal stress was studied using either zymosan, opsonized zymosan (OZ), or phorbol myristate acetate (PMA). A diminished capacity to recover from repeated stimulation was found with aging. When stimulated for a second time 24 hours after the primary stimulation, PM from young animals generated 80% of the initial O2- responses to either zymosan, or OZ. Under the same conditions, PM from senescent mice generated 62% of the initial O2- produced in response to zymosan, and 45% in response to OZ. In both age groups the response to a second PMA stimulation comprised only 10% of the primary response. A considerably diminished capacity to generate O2- was also demonstrated in PM from senescent mice after recovery from exposure to thermal stress. Exposure to 42.5 degrees C for 20 minutes was found to be the threshold temperature for irreversible loss of activity in senescent PM, whereas at this temperature, PM from young animals recovered up to 70% of their O2- generating activity. Since NADPH oxidase and superoxide dismutase activities were only mildly affected by the hyperthermia in all age groups, they could not account for the age-related decline in the recovery from stress. Age-related alterations in signal transduction or receptor alterations could possibly play a primary role in this decline.     

Gr-1+ myeloid cells lacking T cell protein tyrosine phosphatase inhibit lymphocyte proliferation by an IFN-gamma- and nitric oxide-dependent mechanism

The T cell protein tyrosine phosphatase is involved in the immune system regulation, as evidenced by defective function and development of several hemopoietic cell populations in T cell protein tyrosine phosphatase (TC-PTP)-deficient mice. In particular, B and T cell proliferation is greatly inhibited when total splenocytes are stimulated by LPS or anti-CD3 mAb. To define the functional defect of TC-PTP(-/-) lymphocytes, we isolated T and B cells from the spleen of TC-PTP(-/-) mice. We show that the proliferative response of lymphocytes was greatly increased when cultured as a purified population, indicating that an inhibitory population is present in TC-PTP(-/-) spleen. However, TC-PTP(-/-) lymphocytes have a 2- to 3-fold lower proliferation rate compared with TC-PTP(+/+) lymphocytes, suggesting that, as shown previously in embryonic fibroblasts, TC-PTP is involved in the control of cell cycle in lymphocytes. We have characterized phenotypically and functionally the inhibitory population present in the spleen of TC-PTP(-/-) mice. We show that a Gr-1(+)-enriched cell population isolated from TC-PTP(-/-) mice suppresses the CD3-induced proliferation of T cells in coculture in vitro. The specific inhibition of NO synthesis with N(G)-monomethyl-L-arginine.monoacetate restored splenocyte responses, and there is a strict correlation between NO levels and the degree of suppression. Neutralization of IFN-gamma with specific mAb almost completely abolished the inhibitory activity of Gr-1(+) cells and concomitantly high levels of NO secretion. Moreover, inhibition of lymphocyte proliferative responses required cell-cell contact to achieve sufficient levels of NO. These findings demonstrate an important function of TC-PTP in the induction of the NO pathway that mediates inhibition of T cell proliferation.     

Oxygen metabolism of human colostral macrophages: comparison with monocytes and polymorphonuclear leukocytes

Human colostral macrophages stimulated by opsonized zymosan or phorbol myristate acetate (PMA) released superoxide anions (O2-) and hydrogen peroxide (H2O2) with activities comparable to those of monocytes and about one-fourth of those of polymorphonuclear leukocytes (PMNL) of blood. The O2- -forming oxidase in the macrophages stimulated by PMA was dependent on NADPH as an electron donor with an apparent Km value for NADPH of 27.6 +/- 4.0 microM, which is comparable to those obtained for the stimulated monocytes and PMNL of blood. The Vmax was 1.86 +/- 0.33 nmol O2/min/10(6) cells, which is essentially the same as that of monocytes and about half of that of PMNL. p-Chloromercuribenzoate or cetyltrimethylammonium bromide completely inhibited oxidases of all three types of phagocytes. A b-type cytochrome was identified in the macrophages but the concentrations in the macrophages and monocytes were less than half of that in PMNL. These results suggest that the differences in the O2- -forming activities of the three types of phagocytes are quantitative rather than qualitative. The macrophages and monocytes showed very low activities of myeloperoxidase [EC 1.11.1.7] in contrast to PMNL. The activity of beta-glucuronidase [EC 3.2.1.31] in the macrophages was much higher than those of the monocytes and PMNL, but little difference was observed in the activities of lysozyme [EC 3.2.1.17], catalase [EC 1.11.1.6] and superoxide dismutase [EC 1.15.1.1] among the three types of phagocytes examined. Electron micrographs of the macrophages showed little increase of vacuoles upon exposure to PMA, in contrast to the cases of monocytes and PMNL.