Macrophage Nitric Oxide Research Articles

The immunosuppressive effects of chronic morphine treatment are partially dependent on corticosterone and mediated by the μ-opioid receptor

Wild-type and mu-opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the mu-opioid receptor (MOR) in chronic morphine-mediated immunosuppression. We found that although plasma CORT concentrations in CORT infusion (10 mg/kg/day) and morphine-pellet implantation (75 mg) mice were similar (400-450 ng/ml), chronic morphine treatment resulted in a significantly higher (two- to threefold) inhibition of thymic, splenic, and lymph node cellularity; inhibition of thymic-lymphocyte proliferation; inhibition of IL-2 synthesis; and activation of macrophage nitric oxide (NO) production when compared with CORT infusion. In addition, results show that the inhibition of IFN-gamma synthesis and splenic- and lymph node-lymphocyte proliferation and activation of macrophage TNF-alpha and IL-1beta synthesis occurred only with chronic morphine treatment but not with CORT infusion. These morphine effects were abolished in MORKO mice. The role of the sympathetic nervous system on morphine-mediated effects was investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was able to only partially reverse morphine's inhibitory effects. The results clearly show that morphine-induced immunosuppression is mediated by the MOR and that although some functions are amplified in the presence of CORT or sympathetic activation, the inhibition of IFN-gamma synthesis and activation of macrophage-cytokine synthesis is CORT-independent and only partially dependent on sympathetic activation.

Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides

Synthetic 30-mer phosphorothioate (Ps) oligonucleotides (ODN) comprised of single bases (SdA30, SdC30, SdG30, and SdT30) were assessed for their effects on nitric oxide (NO) production by murine bone marrow macrophages (BMMC) and macrophage cell lines J774 and RAW264.7. Pretreatment of these cells with any of the four Ps ODN inhibited NO production induced by CpG ODN, E. coli DNA (EC DNA), or LPS. This inhibition was time- and dose-dependent and was observed even if the Ps ODN were added as long as 12 h after stimulation. As in the case of stimulatory ODN, inhibition was dependent on backbone structure and length. Thus, all four 30-mer, single-base Ps ODN were inhibitory, and only dG30 among phosphodiester ODN was inhibitory. Together, these observations indicate that Ps ODN can inhibit macrophage production of inflammatory mediators, suggesting a role of these compounds as immunomodulatory agents.

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury.

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.

Leaf extracts of Carlowrightia cordifolia induce macrophage nitric oxide production

Carlowrightia cordifolia ( Acanthaceae) is a medicinal plant used in northeastern Mexico as a traditional remedy against inflammation. As tissue release of nitric oxide (NO) has been correlated with both inflammatory and anti-inflammatory processes, the aim of this study was to determine the effect of C. cordifolia leaf extracts on macrophage NO production. Lipopolysaccharide (LPS)-stimulated and non-LPS-stimulated mouse peritoneal macrophages were incubated with aqueous, ethanol, methanol and hexane extracts of C. cordifolia leaves. All extracts inhibited NO release from LPS-stimulated macrophages, with methanol and hexane extracts showing the greatest inhibition. On the other hand, macrophage cultures treated with extracts without LPS-stimulation produced high releases of NO. These unexpected results suggest two different ways by which leaf extracts may act, depending on cell status. On the other hand, data on NO activity in relation to inflammatory/anti-inflammatory auto-regulatory feedback and high concentrations of NO release by non-stimulated macrophages agreed with the hypothesis that NO may have an inhibitory effect in vascular inflammation.

Adrenaline suppression of the macrophage nitric oxide response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor-alpha and interleukin-10.

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (M phi s) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10). Our results showed that NO, TNF-alpha and IL-10 were concurrently produced following in vitro LPS (10 micrograms/ml) stimulation of murine peritoneal M phi s. Adrenaline suppressed both NO and TNF-alpha with concomitant up-regulation of the IL-10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF-alpha was required for NO production, as the TNF-alpha neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL-10 suppressed NO. In order to determine any functional consequence of adrenaline-mediated IL-10 augmentation on NO production, M phi s were stimulated with LPS and specific neutralizing anti-IL-10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10(-8) M) and an irrelevant control antibody had no effect on the adrenaline-mediated inhibition of NO, but anti-IL-10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF-alpha, at a dose range of 1.9-50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL-10 and addition of TNF-alpha abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up-regulation of IL-10 which, in turn, may suppress TNF-alpha that is required for NO production. Finally, we also observed that the M phi-activating cytokine, interferon-gamma (IFN-gamma), attenuated the inhibitory effect of adrenaline on the LPS NO response.

Macrophage nitric oxide synthase associates with cortical actin but is not recruited to phagosomes.

Nitric oxide (NO) produced from inducible NO synthase (iNOS) is an important component of host defense against intracellular pathogens. To understand how phagocytes deliver NO to ingested microorganisms while avoiding cytotoxicity, we set out to study the subcellular localization of iNOS within macrophages following phagocytosis. Confocal microscopy of immunostained cells showed that iNOS was located not only diffusely within cytoplasm but also in vesicles, as well as immediately adjacent to the peripheral cell membrane. This peripheral iNOS colocalized with the cortical actin cytoskeleton and was removed by the actin-depolymerizing drug cytochalasin B. Biochemical fractionation of RAW 264 macrophages showed that 32.75% (+/-5.11%; n = 3) of iNOS was present in a particulate fraction, which cosedimented with low-density cellular vesicles. Following phagocytosis of latex beads, zymosan, immunoglobulin G-coated beads, or complement-coated zymosan, submembranous cortical iNOS was not recruited to phagosomes, nor was there any relocalization of intracellular iNOS. Similarly, following phagocytosis of Salmonella enterica serovar Typhimurium there was no recruitment of iNOS to the Salmonella vacuole at any stage after internalization. NO mediated significant killing of intracellular S. enterica serovar Typhimurium in RAW macrophages treated with lipopolysaccharide and gamma interferon; this was evident 4 h after infection. Although not recruited to phagosomes, iNOS association with the submembranous cortical actin cytoskeleton is ideally suited to deliver NO to microbes in contact with the cell surface and may contribute to early killing of ingested Salmonella.

Suppression of macrophage nitric oxide production by melanoma: mediation by a melanoma-derived product.

The tumour-bearing state is known to induce immune dysfunction that contributes to increased infectious complications and tumour progression. However, the mechanisms underlying this immunosuppression remain unclear. This study investigated in a murine model the effects of melanoma growth on nitric oxide (NO) production by peritoneal macrophages in vivo and in vitro. B16 and K1735 melanoma cells were inoculated subcutaneously into C57BL/6 and C3H/HeN mice, respectively. Stimulated NO production by elicited peritoneal macrophages was examined in control and melanoma- bearing mice. An in vitro system was established to assess the effects of co-culturing melanoma cells (B16 and K1735) or melanoma-conditioned medium with normal peritoneal macrophages on subsequent NO production. NO production was significantly suppressed in macrophages from melanoma-bearing mice. Co-culture of normal macrophages with melanoma cells in a transwell system or with melanoma-conditioned media in vitro reproduced the defects observed in vivo without affecting macrophage viability, pointing to a melanoma-derived product as the basis for the observed suppression of NO production. This inhibition required RNA and protein synthesis and was dose and time dependent. Using inhibition profiles and neutralizing antibodies, it was demonstrated that this melanoma inhibitory activity was distinct from known NO inhibitors. Preliminary characterization attributed this activity to a melanoma-secreted protein moiety.

It's not a mouse, it's a hamster!

A new hamster model for visceral leishmaniasis has been developed that might provide vital insight into the mechanisms of the disease in humans (P.C. Melby et al. J. Immunol. 166, 1912–1920, 2001). The clinical features of Leishmania donovani infection of Syrian hamsters closely resemble human infection; both species experience progressive increases in visceral parasite burden, cachexia, splenomegaly and hypergammaglobulinaemia. The study found that, although a strong Th1-like immune response is initiated in Leishmania-infected hamsters, macrophages in this species fail to produce the effector molecule nitric oxide. The failure of humans to control visceral leishmaniasis is also thought to be because of defective nitric oxide production. In mice, the most commonly used rodent model for leishmaniasis, infection is controlled by macrophage nitric oxide production. SHK

Augmentation of macrophage functions by an aqueous extract isolated from Platycodon grandiflorum

Platycodon grandiflorum has been claimed to have a wide range of health benefits, which include immunostimulation and antitumor activity. The associated biological mechanisms are unclear; however, of the wide diversity of effects, it is believed that their activities may be exerted through several potent effector cells such as macrophages. Therefore, the effects of an aqueous extract from the root of P. grandiflorum (Changkil: CK) on mouse peritoneal macrophage function were investigated. It was found that CK stimulated macrophage proliferation, spreading ability, phagocytosis, cytostatic activity, and nitric oxide production in a dose-dependent manner, and that the production of cytokines such as TNF-α, IL-1β and IL-6 were similarly increased. CK significantly affected secretion at concentrations greater than 10 μg/ml; its maximal effects were at the concentration of 100 μg/ml. Reverse transcription-polymerase chain reaction showed that CK increased the appropriate cytokine mRNAs. These results suggest that CK is a potent enhancer of macrophage function.

Regression of primary hepatocarcinoma in cancer-prone transgenic mice by local interferon-gamma delivery is associated with macrophages recruitment and nitric oxide production.

The clinical potential of tumor therapies must be evaluated using animal models closely resembling human cancers. We investigated the impact of locally delivered interferon-gamma (IFN-gamma) on primary hepatocarcinoma spontaneously developed by T-SV40 transgenic mice. A single intratumor injection of adenovirus IFN-gamma was sufficient enough to induce in vivo production of biologically active IFN-gamma, as assessed by STAT1 activation. IFN-gamma secretion led to the regression of primary tumor, principally by apoptosis of tumor hepatocytes. The lack of T-cells infiltrates in the liver upon treatment excluded a role of a specific immune response. In contrast, indirect pathways may include tumoricidal function of macrophages. Indeed, they were massively recruited in the entire liver under IFN-gamma treatment; transmigration through hepatic blood vessels could be observed and co-localization with damaged hepatocytes was obvious. This correlated with nonparenchymal liver cell iNOS expression and high level of NO in hepatic extracts. Moreover, in vitro experiments showed that NO releasing agents induced cell death of freshly isolated tumor hepatocytes, suggesting that NO could be one of the major effector molecules. Altogether, these observations defined an important role of IFN-gamma in controlling tumor development in a model of primary hepatocarcinoma.

Inhibition by ginkgolides and bilobalide of the production of nitric oxide in macrophages (THP-1) but not in endothelial cells (HUVEC)

Nitric oxide (NO) is a principal mediator in many physiological and pathological processes. NO produced by constitutive nitric oxide synthase in endothelial cells (eNOS) acts as a vasodilator, whereas excess NO production due to elevated expression of inducible nitric oxide synthase (iNOS) may produce cytotoxic effects to cells in the vascular wall. We demonstrated in our previous work that the extract of Ginkgo biloba leaves (EGb) inhibits iNOS-mediated NO production. The objective of the present study was to investigate the effects of several active EGb components on iNOS-mediated NO production in macrophages derived from a human monocytic cell line (THP-1), as well as on eNOS-mediated NO production in human umbilical vein endothelial cells (HUVEC). Ginkgolide A, ginkgolide B, or bilobalide (0.25 to 1.0 μg/mL) caused a 30–65% reduction in the levels of NO metabolites released by THP-1 macrophages after 4 hr of incubation, with a corresponding decrease in iNOS activity. Western immunoblotting analysis coupled with a nuclease protection assay and reverse transcription–polymerase chain reaction revealed a concomitant reduction in the levels of iNOS protein mass and mRNA in ginkgolide A-, ginkgolide B-, or bilobalide-treated macrophages. On the other hand, these compounds did not affect eNOS-mediated NO production or the expression of eNOS protein and mRNA in HUVEC. Taken together, these results suggest that ginkgolide A, ginkgolide B, and bilobalide may contribute to the selective inhibitory effect of EGb on iNOS expression without affecting eNOS-mediated NO production.

The phosphodiesterase inhibitors pentoxifylline and rolipram suppress macrophage activation and nitric oxide production in vitro and in vivo.

We studied the effects of the phosphodiesterase inhibitors pentoxifylline (PTX) and rolipram (ROL) on nitric oxide (NO) production by macrophages and correlated this with cellular cAMP levels. The RAW 264.7 cell line or mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and interferon gamma (IFN gamma), with or without ROL, PTX, cAMP analogues, or Forskolin. In vivo, peritoneal macrophages were stimulated with staphylococcal enterotoxin B with or without administration of ROL. Nitrite levels in culture and the total cellular cAMP levels were measured. ROL and PTX suppressed NO production of LPS/IFN gamma-stimulated macrophages. ROL (IC(50) = 68-74 microM) was about 40 times more potent than PTX (IC(50) = 2.4-2.9 mM). The suppression paralleled increased total cellular cAMP level (EC(50) = 68-72 microM) and was mimicked by other cAMP elevating agents. ROL and PTX suppressed inducible NO synthase at the mRNA level. The inhibition of NO production of macrophages by ROL or PTX could be beneficial in NO-mediated inflammatory and/or autoimmune disorders.

Effect of culture PO2 on macrophage (RAW 264.7) nitric oxide production.

Macrophages are commonly cultured at a PO2 of 149 Torr, but tissue macrophages in vivo live in an environment of much lower oxygen tension. Despite the many potential mechanisms for changes in oxygen tension to influence nitric oxide (NO) synthesis, there have been few reports investigating the effect of PO2 on macrophage NO production. With the use of a culture chamber designed to rigorously control oxygen tension, we investigated the effects of culture PO2 on macrophage NO production, inducible nitric oxide synthase (iNOS) activity, iNOS protein, and tumor necrosis factor production. NO production and iNOS activity were linearly related in the range of 39.4 to 677 Torr, but not in the range of 1.03 to 39.4 Torr. Therefore, results obtained in vitro for the high oxygen tensions commonly used in cell culture were quantitatively and qualitatively different from results obtained in cells cultured at the lower oxygen tensions that more accurately reflect the in vivo environment. The influence of oxygen tension on NO production has implications for cell culture methodology and for the relationship between microcirculatory dysfunction and inflammatory responses in rodent models of sepsis.

Skin Dendritic Cells in Murine Cutaneous Leishmaniasis

Studies of the immunopathogenesis of Leishmania major-induced murine cutaneous leishmaniasis provide a framework for understanding the evolution of L. major infection of skin in humans and the foundation for rationale vaccine design. Experiments in which infection is initiated with "suprap hysiologic" numbers of parasites clearly identify Th-derived type I cytokines as essential participants in macrophage activation and macrophage nitric oxide production as prerequisite for parasite control. Dendritic cells, rather than macrophages, appear to be responsible for L. major-specific Th priming in these studies. Recent studies of murine cutaneous leishmaniasis in a model system in which infection is initiated with lower, more physiologic numbers of parasites confirm many of the important findings obtained in "high dose" inoculation models, but important differences have been noted. The low dose inoculation model should ultimately provide insights into mechanisms that are responsible for dendritic cell recruitment into leishmania lesions, mechanisms that facilitate parasite acquisition by skin dendritic cells and cellular interactions that eventuate in T cell priming and lesion involution.

Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-α and nitric oxide from macrophages via IFN-γ induction

IL-12 is believed to play an important role in cell-mediated immunity against intracellular infection primarily by acting on T and NK cells. Recent evidence has suggested, however, that IL-12 has broader cellular targets than previously thought. In this study, we examined the role of IL-12 in macrophage TNF-alpha and nitric oxide (NO) release by using an in vitro model of intracellular infection. IL-12 alone released relatively little TNF-alpha and NO, whereas live mycobacteria alone released TNF-alpha markedly but little NO from murine alveolar macrophages. However, IL-12 and mycobacteria together enhanced TNF-alpha and NO release synergistically. Because IL-12 and mycobacteria also released IFN-gamma from macrophages synergistically, and exogenous IFN-gamma with mycobacteria enhanced TNF-alpha and NO release synergistically, we examined the role of endogenous IFN-gamma in IL-12/mycobacteria-stimulated macrophage activation. Using macrophages from mice deficient in IFN-gamma, we found that IL-12/mycobacteria-enhanced macrophage TNF-alpha and NO release was mediated through endogenous IFN-gamma. We further demonstrated that IFN-gamma and mycobacteria together had a selective effect on macrophage cytokine release because they released TNF-alpha synergistically but not macrophage chemotactic protein-1 (MCP-1). These findings reveal that IL-12 can activate macrophages potently during intracellular infection, and this activating effect is mediated primarily through its effect on macrophage IFN-gamma release.

Does burn wound excision after thermal injury attenuate subsequent macrophage hyperactivity and immunosuppression?

Studies have shown that cell mediated immunity is suppressed markedly following thermal injury. Macrophages and the activation of an inflammatory cascade that includes interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNFalpha) and PGE2 have been implicated as causative factors. Burn wound excision and grafting is a common clinical practice that decreases patient morbidity and mortality. It is not known, however, if the salutary effects of this procedure are related to modulation of macrophage activity post-burn. Therefore, C57BL/6 female mice were subjected to a third-degree scald burn covering 25% of their total body surface area followed by complete excision and allografting of the injury site at 8, 24, or 72 h post-burn. Splenic macrophage function was assessed 7 days post-burn. Thermal injury without burn excision and grafting significantly increased macrophage TNFalpha, IL-6, nitric oxide, and PGE2 production in response to lipopolysaccharide stimulation, whereas IL-1beta production was not increased. Burn wound excision and grafting normalized TNFalpha production to sham levels, independent of when post-burn the procedure was conducted. In contrast, the elevated production of other inflammatory mediators (IL-1beta, IL-6, nitric oxide, PGE2) post-burn was unaffected by burn wound excision and grafting. Moreover, splenic T-lymphocyte proliferation was also suppressed at 7 days post-burn and was not improved by burn wound excision and grafting. These results, therefore, suggest that the beneficial effects of burn wound excision and grafting are likely to be related to the normalization of macrophage TNFalpha production as well as the maintenance of skin barrier function.

INTERACTION OF NITRIC OXIDE AND TRANSFORMING GROWTH FACTOR-β1 INDUCED BY ANGIOTENSIN II AND MECHANICAL STRETCH IN RAT RENAL TUBULAR EPITHELIAL CELLS

Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch. NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch. These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.

Role of catalase in Campylobacter jejuni intracellular survival.

The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.

The role of CD40L in T cell-dependent nitric oxide production by murine macrophages

Upon activation, T cells express CD40L, a member of the TNF cytokine superfamily, which serves as a ligand for CD40 on antigen presenting cells, including dendritic cells, B cells, and macrophages. While initial studies on the function of CD40 focused its role in the regulation of B cell activation, more recent studies have indicated that CD40 ligation may be critical for the initiation of T cell-dependent macrophage activation, including stimulation of nitric oxide production. However, the relative contribution of the CD40 pathway in macrophage nitric oxide production during T-dependent immune responses remains unclear. We have found that while CD40 ligation of macrophages can stimulate nitric oxide production, disruption of CD40 signaling during a T cell-mediated alloimmune response has no appreciable effect on nitric oxide production. If the T cell alloimmune response is restricted to CD4 cells, CD40L blockade has only a minimal effect on nitric oxide production. Rather, IFNγ, produced by alloactivated T cells, seems to be a necessary ‘first’ signal for nitric oxide production, while TNFα and CD40L each provide independent ‘second’ signals. Finally, we demonstrate that CD40L stimulates macrophage NO production independent from autocrine TNFα stimulation. These results suggest that macrophage nitric oxide production during a T-dependent immune response requires IFNγ production by CD4 cells whereas TNFα and CD40L can each provide important functionally overlapping ‘second’ signals to costimulate nitric oxide production, though neither is required.

Evaluation of Nitric Oxide Synthase Activity and Inhibition Kinetics by Chemiluminescence

The binding affinity (KI) and inactivation rate (kinact) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.