Granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces toxoplasmastatic activity of human monocytes via induction of prostaglandin E2 (PGE2).

In this study we investigated the effect of human GM-CSF on the toxoplasmastatic activity and release of H2O2 and PGE2 by human monocytes. Incubation of monocytes from healthy controls with GM-CSF resulted in a dose-dependent reduction of toxoplasmastatic activity and a decrease in H2O2 production. Furthermore GM-CSF-treated monocytes released more PGE2 than untreated cells. To investigate the role of PGE2 in the reduced toxoplasmastatic activity of GM-CSF-treated monocytes, these cells were incubated with indomethacin. This resulted in a reduction of PGE2 release and restoration of toxoplasmastatic activity of monocytes treated with GM-CSF. GM-CSF reduces the toxoplasmastatic activity of monocytes via production of PGE2.     

Endogenous tumor necrosis factor alpha is required for enhanced antimicrobial activity against Toxoplasma gondii and Listeria monocytogenes in recombinant gamma interferon-treated mice.

In vitro studies have shown that macrophages stimulated with recombinant gamma interferon (rIFN-gamma) produce tumor necrosis factor alpha (TNF-alpha), which in an autocrine fashion activates these cells. The aim of the present study was to determine whether endogenously formed TNF-alpha also is required for rIFN-gamma-induced macrophage activation and enhanced antimicrobial activity in vivo. After an intraperitoneal injection of rIFN-gamma into CBA/J mice, their peritoneal macrophages released enhanced amounts of NO2- and inhibited the intracellular proliferation of Toxoplasma gondii. Injection of neutralizing antibodies against TNF-alpha simultaneously with the rIFN-gamma completely inhibited both the release of NO2- by macrophages and their toxoplasmastatic activity. Similar results were observed after intraperitoneal injection of a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, together with rIFN-gamma, demonstrating that in vivo L-arginine-derived reactive nitrogen intermediates are essential for the induction of toxoplasmastatic activity. Intravenous injection of rIFN-gamma inhibited the growth of Listeria monocytogenes in the livers and spleens of mice; this effect was abrogated by antibodies against TNF-alpha. Intravenous injection of a large dose of rTNF-alpha resulted in a decrease in the number of bacteria in the liver and spleen, but an injection of rIFN-gamma and rTNF-alpha did not result in enhanced inhibition of the proliferation of L. monocytogenes. Together, the results of the present study are the first to demonstrate that endogenous TNF-alpha is required in vivo for the expression of macrophage activation with respect to the release of reactive nitrogen intermediates and toxoplasmastatic activity and for enhanced listericidal activity in the livers and spleens of mice stimulated with rIFN-gamma.     

Granulocyte-macrophage colony-stimulating factor is not involved in production of reactive nitrogen intermediates by or toxoplasmastatic activity of gamma interferon-activated murine macrophages.

The induction of reactive nitrogen intermediates (RNI) and toxoplasmastatic activity of murine macrophages by recombinant gamma interferon (rIFN-gamma) is mediated by an autocrine pathway involving tumor necrosis factor alpha (TNF-alpha). To investigate whether cytokines other than TNF-alpha play a role in the activation of these effector functions, granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied. Recombinant GM-CSF (rGM-CSF) could stimulate peritoneal macrophages, since this cytokine stimulated the production of prostaglandin E2 by these cells. However, rGM-CSF did not induce either the release of RNI by or the toxoplasmastatic activity of macrophages. rGM-CSF in combination with various concentrations of rIFN-gamma did not enhance these effector functions more than rIFN-gamma alone. Furthermore, neutralization of endogenously produced GM-CSF by monoclonal antibodies did not affect the release of RNI by or the toxoplasmastatic activity of rIFN-gamma-activated macrophages. Together these results indicate that GM-CSF is not involved in RNI production by and toxoplasmastatic activity of IFN-gamma-activated murine macrophages.     

Incorporation of recombinant gamma interferon into liposomes enhances its ability to induce peritoneal macrophage antitoxoplasma activity.

In this study we compared the ability of free- and liposome-incorporated murine recombinant gamma interferon (rIFN-gamma) to enhance peritoneal macrophage H2O2 release and antitoxoplasma activity in vitro. rIFN-gamma was efficiently (37 to 47%) incorporated into multilamellar vesicles composed of phosphatidylglycerol/cholesterol in a 2:1 molar ratio. The amount of rIFN-gamma incorporated into multilamellar vesicles and added to macrophages (0.1 to 1,000 U/ml) was quantitated with [3H]rIFN-gamma. The concentration of liposomal rIFN-gamma required to enhance macrophage H2O2 release (1 U/ml) and maximally inhibit Toxoplasma gondii growth (10 U/ml) was one-tenth the concentration required for free rIFN-gamma (10 and 100 U/ml, respectively). This increase in potency was observed in both thioglycolate-elicited and resident peritoneal macrophages. Control liposomes containing encapsulated buffer had no effect on the potency of free rIFN-gamma. The duration of macrophage activation induced by 24 h of liposomal rIFN-gamma treatment was also considerably longer than that induced by free rIFN-gamma (2 days versus less than 1 day). These data indicate that liposomal rIFN-gamma is more active than free rIFN-gamma as an inducer of macrophage microbicidal properties in vitro. This enhanced activity, combined with the potential for selective delivery of liposomal rIFN-gamma to phagocytic cells in vivo, may improve the therapeutic efficacy of rIFN-gamma in infections characterized by parasitization of phagocytes.     

Microbicidal activities of Salmonella typhimurium- and interferon-gamma-activated mouse peritoneal macrophages

Activation of mouse peritoneal macrophages during infection of mice by various facultative intracellular bacteria and after intravenous injection of recombinant interferon-gamma (rIFN-gamma) was studied. Macrophage activation was demonstrated on the basis of three different criteria, i.e. inhibition of Toxoplasma gondii proliferation, enhanced release of H2O2 and increased expression of Ia antigen. Macrophages activated during an infection with Salmonella typhimurium showed no enhanced salmonellacidal or listericidal activity relative to control macrophages, whereas Listeria-activated macrophages killed Listeria but not Salmonella faster than control macrophages. The rate of proliferation of Salmonella in spleen and liver of activated mice was comparable to the proliferation in the organs of control mice. rIFN-gamma-activated macrophages displayed neither an enhanced salmonellacidal nor an enhanced listericidal activity. When high numbers of Listeria were injected intravenously the proliferation in spleen and liver of rIFN-gamma-treated and control mice was similar. The proliferation of Listeria in the liver of rIFN-gamma-treated mice was less than in control mice when 1 LD50 or lower numbers of bacteria were injected. It is concluded that peritoneal macrophages become activated during infections of mice with various intracellular pathogens. However, these activated macrophages do not show enhanced bactericidal activity against all bacteria. Furthermore, rIFN-gamma is not sufficient to enhance the listericidal activity of macrophages.     

Peripheral blood monocytes derived from HIV+ individuals mediate antibody-dependent cellular cytotoxicity (ADCC)

Monocytes/macrophages serve a number of immunologic functions and play a major role in the host defense against infection. Abnormal functions of monocytes have been reported in AIDS and HIV+ individuals. A recent report from our laboratory demonstrated that peripheral blood monocytes (PBM) derived from AIDS patients were de novo "activated" as assessed by direct cell-mediated cytotoxicity (CMC) and secretion of cytotoxic factors and tumor necrosis factor-alpha (TNF alpha). Thus, both the direct cytotoxicity as well as the antibody-dependent cellular cytotoxicity (ADCC) exerted by the monocytes may contribute to the destruction of HIV-infected/coated cells and the immunopathogenesis of AIDS. The present study investigated the ability of HIV+ PBM to mediate ADCC against antibody-coated target cells in an 18-hr 51Cr release assay. Initial studies examined ADCC using a macrophage resistant target Raji and rabbit anti-Raji serum. The results show that the majority of PBM from HIV+ individuals mediate ADCC activity while the majority of PBM from normal healthy controls was not cytotoxic. While activation of PBM with recombinant interferon-gamma (rIFN-gamma) enhances the ADCC activity of normal PBM, treatment of HIV+ PBM with IFN-gamma resulted in significant enhancement of ADCC. Both untreated and treated PBM from HIV+ individuals had significantly higher ADCC than PBM from normal individuals. Of interest, a significant ADCC activity was found by PBM derived from two HIV- high risk individuals whether untreated or treated with rIFN-gamma. The ADCC results with RAJI target cells prompted us to investigate whether ADCC can also be obtained using HIV-infected T4+ cells. We selected a macrophage and TNF resistant T4+ CEM cell line as target for ADCC. The target was coated with inactivated HIV and pooled human anti-HIV serum was used. Studies with a few HIV+ individuals demonstrate that significant ADCC is obtained with PBM from HIV+ individuals but little or no ADCC by normal PBM and the ADCC was specific for HIV. The ADCC was also significantly enhanced by treatment of PBM with rIFN-gamma. The results of this study clearly indicate that PBM from HIV+ individuals are endowed with the capacity to mediate ADCC against HIV-infected/coated cells and thus, we postulate that PBM may play a direct role in vivo in lysis or suppression of HIV-coated/infected cells and in the pathogenesis of AIDS.     

Reactive oxygen intermediates, nitrite and IFN-gamma in Indian visceral leishmaniasis.

Reactive oxygen intermediates (ROI), nitrite and interferon-gamma (IFN-gamma) production were investigated at different times during treatment in 10 patients with visceral leishmaniasis (VL). Hydrogen peroxide (H2O2), superoxide (O2-) and IFN-gamma production by cultured monocytes from patients with active VL were significantly lower compared with the healthy controls. In contrast, nitrite levels in the supernatants from monocyte cultures of VL patients were comparable to healthy controls and increased significantly during antileishmanial therapy. On day 20 of treatment, a significant increase in the release of H2O2, O2- and IFN-gamma was observed. However, at follow-up, 4 months after the end of treatment, the production of H2O2, O2-, IFN-gamma and nitrite had declined significantly. Thus, the impairment in hydrogen peroxide and superoxide production suggests that down-regulation of these mediators may be involved in the reduced killing of parasites by monocytes of active VL patients. Furthermore, the monocytes regained respiratory burst activity as the antileishmanial therapy progressed, suggesting that an immune-based mechanism is involved in successful drug therapy.     

Nitric Oxide Mediates the Toxoplasmastatic Activity of Murine Microglial Cells in vitro

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice.

Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gM) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-aL) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-aL in combination with a non-activating concentration of rIFN-gM released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-aL inhibited the release of RNI by rIFN-gM activated macrophages.

In summary, the present results show that activation of microglial cells by rIFN-gM and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.     

Hydrocortisone Treatment of BCG-Infected Mice Impairs the Activation and Enhancement of Antimicrobial Activity of Peritoneal Macrophages

The present study concerns the effect of hydrocortisone (HC) on the effector functions of Bacillus Calmette Guerin-purified protein derivative (BCG-PPD)-activated macrophages. Such activated macrophages release greater amounts of H2O2 and NO2-, inhibit the intracellular proliferation of T. gondii and kill L. monocytogenes more efficiently than resident macrophages. This activation was not fully expressed by macrophages from BCG-activated mice that had received a subcutaneous injection of HC 2 days before intraperitoneal injection of PPD, since the inhibition of the intracellular proliferation of T. gondii, the release of NO2- and the rate of intracellular killing of L. monocytogenes were lower than in macrophages from BCG-PPD-activated mice. However, treatment with HC did not impair the release of H2O2 by BCG-PPD-activated macrophages. The results show that the treatment of infected mice with HC inhibits their ability to develop adequate intracellular microbicidal mechanisms.     

Nitric Oxide Mediates the Toxoplasmastatic Activity of Murine Microglial Cells in vitro

Toxoplasma gondii (T. gondii), an opportunistic protozoan, is an important cause of central nervous system (CNS) infections in immunosuppressed patients. The present study focused on the interaction between T. gondii and microglial cells from the brain of neonatal Balb/c mice.

Preincubation of the murine microglial cells with recombinant interferon-gamma (rIFN-gM) and lipopolysaccharide (LPS) induced significant inhibition of T. gondii replication in a dose dependent manner. This antiparasitic effect in microglial cells was correlated with the induction of the L-arginine-dependent generation of reactive nitrogen intermediates (RNIs). Tumor necrosis factor-alpha (TNF-aL) was also involved in the toxoplasmastatic activity. Microglial cells incubated with recombinant TNF-aL in combination with a non-activating concentration of rIFN-gM released substantial amount of RNIs. Neutralizing antibodies against mouse TNF-aL inhibited the release of RNI by rIFN-gM activated macrophages.

In summary, the present results show that activation of microglial cells by rIFN-gM and LPS induce the production of nitric oxide (NO) by these cells via an L-arginine dependent pathway. NO appears to be the effector molecule mediating the toxoplasmastatic effects in these cells.     

Intravenous injection of interferon-gamma inhibits the proliferation of Listeria monocytogenes in the liver but not in the spleen and peritoneal cavity.

In the present study the effects of intravenous administration of recombinant interferon-gamma (IFN-gamma) on both the proliferation of Listeria monocytogenes in the liver and spleen of mice and the listericidal activity of their peritoneal macrophages were investigated. A single intravenous injection of 1 x 10(6) U or three injections of 2 x 10(5) U recombinant IFN-gamma (rIFN-gamma) induced optimal activation of resident and exudate peritoneal macrophages, as judged by their ability to inhibit the intracellular proliferation of Toxoplasma gondii and their enhanced release of H2O2 and NO2-. The rate of intracellular killing of L. monocytogenes by the rIFN-gamma-activated resident and exudate macrophages was not higher than that by resident macrophages. Addition of 10 ng lipopolysaccharides (LPS) to the rIFN-gamma also did not enhance the bactericidal activity of the activated peritoneal macrophages. The decrease in the number of L. monocytogenes in the peritoneal cavity of mice that had received an i.p. injection of 1 x 10(4) U rIFN-gamma was similar to that in control mice. Intravenous administration of 1 x 10(5) rIFN-gamma activated cells in the liver, as indicated by the increased expression of Ia antigen, and reduced the rate of proliferation of L. monocytogenes in the liver relative to that in control mice when 0.1 LD50 or 1 LD50 L. monocytogenes were injected. However, when 10 LD50 L. monocytogenes were administered there was no effect on their proliferation. The number of L. monocytogenes found initially in the spleen of rIFN-gamma-treated mice was 20-30% of that in the spleen of control mice, but the rate of proliferation of L. monocytogenes was not reduced. These divergent results for the proliferation of L. monocytogenes in the liver, spleen and peritoneal cavity indicate that cells other than macrophages and/or as yet unknown local factors play an important role in the listericidal activity.     

Class II major histocompatibility complex antigen expression on unstimulated and gamma-interferon stimulated monocytes from patients with multiple sclerosis, rheumatoid arthritis and normal controls.

HLA-DR, HLA-DQ, and HLA-DP antigen expression was assessed by immunofluorescent flow cytometry on monocytes from 19 patients with active multiple sclerosis (MS), 19 with inactive MS, 7 patients with early active rheumatoid arthritis (RA), and 19 normal controls. Percentage positivity and median channel fluorescence (MCF) were determined after separation of the monocytes (TO) and following 48 h culture with (T48 + IFN) and without (T48) recombinant gamma interferon (rIFN-gamma). The percentage positivity of the cells was normal at TO for all groups of patients for each of the HLA types but statistically significantly increased above normal, on monocytes from patients with inactive MS, after culture with rIFN-gamma. At TO, the MCF values for HLA-DQ, and HLA-DP were statistically significantly increased above normal on monocytes from patients with active MS indicating some pre-programming of the cells in vivo. After culture, when the carry-over from baseline TO values was eliminated, the increment in MCF for HLA-DR, on monocytes from patients with inactive MS, was statistically significantly lower than normal in the non-gamma-IFN cultures but was normal in the presence of rIFN-gamma. Conversely, the increment in MCF for HLA-DP on monocytes from patients with active MS was significantly lower than normal after culture with rIFN-gamma. Therefore, the stimulation required to increase antigen density on cells already expressing antigen may be different to that required to stimulate de novo expression on negative cells. Both systems appear to be abnormal in MS, possibly reflecting differences in disease activity, while only one system appears abnormal in RA.     

Mycobacterial 65-kilodalton heat shock protein induces tumor necrosis factor alpha and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages.

The 65-kDa heat shock protein (Hsp65) is supposed to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. These effects are thought to result mainly from an Hsp65-specific T-lymphocyte-mediated immune response that recognizes conserved epitopes. The aim of the present study was to assess whether mycobacterial Hsp65 has a direct effect on resident murine peritoneal macrophages, independent of Hsp65-sensitized T lymphocytes. Exposure of peritoneal macrophages from naive C57BL/6 mice to the mycobacterial Hsp65 in vitro induced an enhanced release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6. These cells also produced large amounts of reactive nitrogen intermediates (RNI) and inhibited the intracellular proliferation of Toxoplasma gondii. Small amounts of gamma interferon acted synergistically with Hsp65. Thus, exposure of murine macrophages to Hsp65 results in activation of these cells. The acquisition of these characteristics by peritoneal macrophages occurred in the absence of sensitized T lymphocytes. Addition of anti-TNF-alpha antiserum resulted in an attenuation of the Hsp65-induced release of RNI and toxoplasmastatic activity, indicating that endogenous TNF-alpha is involved in the Hsp65-induced macrophage activation. The conclusion of this study is that in vitro exposure of peritoneal macrophages to the mycobacterial Hsp65 induces the release of proinflammatory cytokines and RNI and results in inhibition of the intracellular proliferation of T. gondii. These effects on murine macrophages occur independently of Hsp65-specific T lymphocytes. The proinflammatory effect of Hsp65 demonstrated in this study suggests that this heat shock protein may play a role in the initiation of inflammation that adds to a non-species-specific resistance in the early stages of infections.     

Defective monocyte function in acquired immune deficiency syndrome (AIDS): Evidence from a monocyte-dependent T-cell proliferative system

T-cell proliferative responses to the mitogenic monoclonal antibody anti-Leu 4 were assessed in healthy controls, lymphadenopathy syndrome (LAS) patients, and acquired immune deficiency syndrome (AIDS) patients. While 19% of the control group showed low anti-Leu 4 responses (<12,000 cpm), 60% of the LAS patients, 71% of the AIDS-opportunistic infection patients, and 50% of the AIDS-Kaposi's sarcoma patients showed low responses. T-cell responsiveness in healthy low responders was greatly enhanced by the addition of monocytes from an anti-Leu 4 high responder (responder monocytes). We therefore sought to determine if the low-responder state in LAS and AIDS patients was also mediated by monocytes and, thus, correctable by the addition of responder monocytes. In the LAS low-responder group, the level of enhancement by healthy responder monocytes was similar to that observed for the healthy low-responder group. In the AIDS low-responder group, however, the level of enhancement was significantly lower than that observed in the healthy low-responder and LAS low-responder groups. These findings suggest that impaired proliferation to anti-Leu 4 in LAS patients may be due to a monocyte defect similar to the monocyte defect responsible for low anti-Leu 4 responses in healthy controls. AIDS patients, however, show additional defects in anti-Leu 4-induced proliferation that are not fully corrected by the addition of responder monocytes.     

Microbicidal activity of monocyte derived macrophages in AIDS and related disorders.

We have examined the ability of monocyte-derived macrophages from patients with AIDS and other HIV-related disorders to kill the intracellular pathogen Toxoplasma gondii. We have also examined the capacity of peripheral blood mononuclear cells from these patients to produce macrophage-activating and other lymphokines. The capacity to produce interleukin 2 and gamma interferon decreases from controls through asymptomatic seropositive subjects and lymphadenopathy groups A (benign) and B (prodromal) to AIDS. The decrease did not correlate precisely with the decrease in CD4+ cells in these patients. Monocyte-derived macrophages from asymptomatic HIV-infected subjects and lymphadenopathy patients showed a decreased ability to kill T. gondii after activation with recombinant gamma interferon; paradoxically, this was most striking for PGL group A. The defect was largely overcome by using Concanavalin A stimulated autologous supernatants. It was notable that macrophages from AIDS patients showed normal killing with recombinant gamma interferon, but that the supernatants from AIDS patients had reduced activity with normal macrophages. These studies confirm that functional defects of both lymphocytes and macrophages are found in HIV-infected subjects; they serve to emphasize the heterogeneity of the clinical and biological responses to this retrovirus, responses which have important implications in the pathogenesis and treatment of the immunodeficiency.     

Generation of superoxide anion by alveolar macrophages in sarcoidosis: evidence for the activation of the oxygen metabolism in patients with high-intensity alveolitis.

We studied superoxide anion (O2) generation by alveolar macrophages (AM) isolated from bronchoalveolar lavages (BAL) of patients with sarcoidosis, and assayed immediately after the isolation or after maintenance in culture for 2 days. In assays of cells freshly isolated from BAL, AM of patients with active sarcoidosis with a high-intensity lymphocytic alveolitis produced more O2- in response to phorbol myristate acetate than AM of patients with inactive sarcoidosis. Also, after 2 days of cultivation sarcoid AM were heterogeneous in their capability to metabolize oxygen, although both AM of active and inactive sarcoid patients produced higher amounts of O2- than AM of healthy subjects. In vitro treatment with recombinant interferon-gamma (rIFN-gamma) caused an enhancement of the capability of AM of inactive sarcoid patients to produce O2- in response to PMA. AM of patients with active sarcoidosis did not respond to rIFN-gamma when they already produced O2- vigorously. However, they became sensitive to the activating effect of rIFN-gamma after the down-modulation of their capability to produce O2-, that occurred upon prolonged cultivation. Monocytes isolated from blood of sarcoid patients and assayed immediately or after different times of cultivation did not produce more O2- than control monocytes and monocyte-derived macrophages, thus indicating that the activation of AM in sarcoidosis is likely a local phenomenon. These studies strengthen the notion that T lymphocyte-macrophage interaction is a critical event in the pathogenesis of sarcoidosis and establish that the enhanced capability to metabolize oxygen to highly reactive intermediates by AM is one of the consequence of this interaction.     

Macrophage oxidative metabolism and intracellular Toxoplasma gondii

We explored the mechanisms by which Toxoplasma gondii avoids destruction by the oxidative metabolism of normal macrophages. Unelicited murine peritoneal macrophages were infected with T. gondii and exposed to different experimental conditions. As endpoints we used measurement of hydrogen peroxide (H2O2) release and intracellular reduction of nitroblue tetrazolium dye (NBT). Three main observations were made. Firstly, different T. gondii preparations (live or dead, opsonized or not) failed to trigger the respiratory burst. Combined challenges also showed that a primary T. gondii infection was able to block H2O2 release triggered by heat-killed (HK)-Candida albicans. The H2O2 release, however, once triggered by HK-C. albicans, was not inhibited by a subsequent challenge with T. gondii. Secondly, when a respiratory burst was obtained in T. gondii-infected macrophages--for instance by stimulation with phorbol myristate acetate (PMA)--the toxic oxygen metabolites (as determined by the NBT reduction test) did not seem to reach the vacuoles containing the parasite. Thirdly, when a respiratory burst occurred in T. gondii-infected macrophages, the intracellular development of T. gondii did not seem to be affected. In conclusion, we hypothesize that T. gondii is not damaged by the macrophage oxidative metabolism because the parasite fails to encounter toxic oxygen metabolites. The killing of intracellular T. gondii, as it is commonly observed in activated macrophages, does not appear oxygen-dependent.     

Effects of gamma interferon and nitric oxide on the interaction of Mycobacterium avium subsp. paratuberculosis with bovine monocytes.

In this study, we examined the effects of recombinant bovine gamma interferon (rIFN-gamma) and nitric oxide (NO) on the interaction of M. avium subsp. paratuberculosis with bovine monocytes. Monocytes pretreated with rIFN-gamma exhibited slightly increased phagocytosis of M. avium subsp. paratuberculosis and modest inhibition of the intracellular growth of this microorganism. The number of viable intracellular bacilli decreased earlier in rIFN-gamma-pretreated monocytes than in control monocytes. After infection with M. avium subsp. paratuberculosis, NO was not constitutively released, but NO release from infected monocytes was induced by treatment with rIFN-gamma or with rIFN-gamma and lipopolysaccharide (LPS). Release of nitric oxide was inhibited by addition of N(G)-monomethyl-L-arginine; however, inhibition of nitric oxide did not alter the pattern of intracellular survival of M. avium subsp. paratuberculosis in rIFN-gamma-treated bovine monocytes. Although chemically generated nitric oxide killed M. avium subsp. paratuberculosis in a cell-free system in vitro, the amount of nitric oxide required was far greater than that released from infected monocytes stimulated with rIFN-gamma and LPS. Our data suggest that rIFN-gamma activates M. avium subsp. paratuberculosis-infected bovine monocytes to release nitric oxide but only modestly increases antimycobacterial activity of monocytes against this organism. This may be due, in part, to the fact that the amount of nitric oxide produced by rIFN-gamma-activated bovine monocytes is insufficient to kill intracellular M. avium subsp. paratuberculosis bacilli in vitro.     

Reduced oxidative burst responses in monocytes and monocyte-derived macrophages from HIV-infected subjects.

Oxidative burst responses of monocytes and monocyte-derived macrophages (MDM) were studied in 40 subjects with HIV infection of different clinical stages. Oxidative burst was assessed as reduction of nitroblue tetrazolium (NBT) with or without stimulants. Results were determined as oxidative burst responses per cell and as a stimulatory ratio between stimulated and unstimulated NBT reduction. Cells from 12 HIV-seronegative homosexual men and 38 blood donors served as control groups. In patients with asymptomatic HIV infection, monocyte oxidative burst responses were reduced compared with the blood donors. In MDM from the same patients, stimulatory ratios were reduced. In AIDS patients, stimulatory ratios of both monocytes and MDM were reduced compared with controls. In contrast to the progressive deterioration of CD4+ lymphocyte counts as well as other immune functions in HIV infection, monocyte oxidative burst responses are impaired already in the asymptomatic phase of the infection, almost to the same extent as in patients with AIDS.