Killing Activity Of Macrophages Research Articles

Vaccination Route That Induces Transforming Growth Factor β Production Fails To Elicit Protective Immunity againstLeishmania donovaniInfection

BALB/c mice immunized intraperitoneally (i.p.) and intravenously (i.v.) with Leishmania donovani promastigote membrane antigens (LAg), either free or encapsulated in liposomes, were protected against challenge infection with L. donovani, whereas mice immunized by the subcutaneous (s.c.) and intramuscular routes were not protected. Protected mice showed strong parasite resistance in both the liver and spleen, along with enhanced immunoglobulin G2a and delayed-type hypersensitivity responses. Again, mice vaccinated through the i.p. and i.v. routes showed high levels of NO production after challenge infection. s.c. vaccination resulted in an increased capacity of the spleen cells to produce prechallenge transforming growth factor beta (TGF-beta) levels during the in vitro antigen recall response, whereas i.p. immunization induced production of prechallenge gamma interferon, interleukin-12 (IL-12), and IL-4 levels, with a Th1 bias. Exposure to antigen-stimulated splenocyte supernatants of i.p. but not s.c. immunized mice activated macrophages for in vitro parasite killing. As an enhanced level of TGF-beta was detected in supernatants from unprotected s.c. immunized mice, neutralization by anti-TGF-beta antibody enhanced in vitro macrophage killing activity. The suppressive role of this cytokine was evaluated in vivo by vaccination with liposomal LAg and anti-TGF-beta antibody. Upon parasite challenge, these animals showed significant protection in both the liver and spleen. Moreover, the addition of recombinant TGF-beta in splenocyte supernatants of i.p. immunized mice in vitro as well as in vivo inhibited the protective ability of the macrophages by the i.p. route. Thus, the induction of high prechallenge TGF-beta limits the efficacy of vaccination by routes that are nonprotective.

Candida albicans cell surface superoxide dismutases degrade host‐derived reactive oxygen species to escape innate immune surveillance

Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real-time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro. ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro. The reduced viability of sod5Δ/Δ and sod4Δ/Δsod5Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox−/− mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans, and perhaps many other microbial pathogens, can evade host immune surveillance in vivo.

The dyspeptic macrophage 30 years later: An update in the pathogenesis of Crohn's disease

Alterations in autophagy leading to a defective intracellular response to low-level invasive bacteria are considered a major recent advance in the pathogenesis of Crohn's disease. A genome-wide association study has shown an association of Crohn's disease with the autophagy related 16-like 1 gene. A second autophagy gene, the immunity-related Guanosine triphosfatase, has also been found to be significantly associated with Crohn's disease. The enteric flora of Crohn's disease patients includes, more commonly than controls, strains of adherent/invasive E. coli. The high level of adherent/invasive E. coli colonizing the intestinal mucosa of patients with Crohn's disease strongly suggests that it may play an important role in the aetiopathogenesis of the disease. E. coli strains are able to cross the mucosal barrier, survive within macrophages and induce the secretion of TNFalpha and the formation of granuloma. Recently it has been clearly shown that Crohn's disease patients have a defective mucosal macrophage killing activity resulting in increased exposure to commensal bacteria and activation of T cells. However, the hypothesis of macrophages dysfunction in the pathogenesis of Crohn's disease was already suggested in 1977 by M. Ward, who introduced the concept of the "dyspeptic macrophage", consisting of an inability to degrade a variety of phagocytosed normal gut dietary and microbial luminal constituents. Defective autophagy and dyspeptic macrophages seems therefore indicate the same pathogenetic mechanism. What is really new is the demonstration that this impaired macrophage function is genetically determined.

Role of RHOB in the antiproliferative effect of glucocorticoid receptor on macrophage RAW264.7 cells

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(-) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression of Rhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(-) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation of Rhob in RAW-GR(-) and RAW264.7 cells by transfection with wild-type Rhob expression vector (Rhob-wt) or constitutively activated Rhob plasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression of Rhob by RNA interference technique or the activity of Rhob by transfection with dominant negative Rhob mutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(-) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate that Rhob plays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR-->Akt-->Rhob signaling and Rhob negatively regulates the proliferation of RAW264.7 cells.

Immunomodulating Effect of Methisoprinol on the Pronephros Macrophage and Lymphocyte Activity after Suppression Induced by Infectious Haematopoietic Necrosis Virus (IHNV) in Rainbow Trout (Oncorhynchus mykiss)

The purpose of this in vitro study was to determine the influence of methisoprinol on the activity of pronephros macrophages and lymphocytes after suppression induced by infectious haematopoietic necrosis virus (IHNV). For this study IHNV-free rainbow trout were used. Pronephros from 20 fish were removed and single leukocyte suspensions were separated. The IHNV significantly (P < 0.05) decreased the respiratory burst activity and potential killing activity of pronephros macrophages and proliferative response of lymphocytes stimulated by mitogens ConA and LPS. The results of our in vitro study showed that methisoprinol at a concentration of 50 mg/ml modulated (restored) the metabolic and potential killing activity of macrophages and proliferative response of lymphocytes suppressed by IHNV.

Constitutive acid sphingomyelinase enhances early and late macrophage killing of Salmonella enterica serovar Typhimurium.

We have identified acid sphingomyelinase (ASM) as an important player in the early and late anti-Salmonella activity of macrophages. A functional ASM participated in the killing activity of macrophages against wild-type Salmonella enterica serovar Typhimurium. The role of ASM in early macrophage killing of Salmonella appears to be linked to an active NADPH phagocyte oxidase enzymatic complex, since the flavoprotein inhibitor diphenyleneiodonium not only blocked a productive respiratory burst but also abrogated the survival advantage of Salmonella in macrophages lacking ASM. Lack of ASM activity also increased the intracellular survival of an isogenic DeltaspiC::FRT Salmonella strain deficient in a translocator and effector of the Salmonella pathogenicity island 2 (SPI2) type III secretion system, suggesting that the antimicrobial activity associated with ASM is manifested regardless of the SPI2 status of the bacteria. Constitutively expressed ASM is responsible for the role that this lipid-metabolizing hydrolase plays in the innate host defense of macrophages against Salmonella. Accordingly, the ASM activity and intracellular concentration and composition of ceramide, gangliosides, and neutral sphingolipids did not increase upon Salmonella infection. Salmonella triggered, nonetheless, a significant increase in the secreted fraction of ASM. Collectively, these findings have elucidated a novel role for constitutive ASM in the anti-Salmonella activity of murine macrophages.

Vitamin D receptor signaling contributes to susceptibility to infection withLeishmania major

We have previously reported that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) can selectively suppress key functions of interferon-gamma (IFN-gamma) activated macrophages. To further explore this mechanism for its relevance in vivo, we investigated an infection model that crucially depends on the function of IFN-gamma activated macrophages, the infection with the intracellular protozoan Leishmania major. 1Alpha,25(OH)2D3 treatment of L. major infected macrophages demonstrated a vitamin D receptor (Vdr) dependent inhibition of macrophage killing activity. Further analysis showed that this was a result of decreased production of nitric oxide by 1alpha,25(OH)2D3-treated macrophages due to Vdr-dependent up-regulation of arginase 1 expression, which overrides NO production by Nos2. When analyzing the course of infection in vivo, we found that Vdr-knockout (Vdr-KO) mice were more resistant to L. major infection than their wild-type littermates. This result is in agreement with an inhibitory influence of 1alpha,25(OH)2D3 on the macrophage mediated host defense. Further investigation showed that Vdr-KO mice developed an unaltered T helper cell type 1 (Th1) response on infection as indicated by normal production of IFN-gamma by CD4+ and CD8+ T cells. Therefore, we propose that the absence of 1alpha,25(OH)2D3-mediated inhibition of macrophage microbicidal activity in Vdr-KO mice results in increased resistance to Leishmania infection.

Inhibitor κB and Nuclear Factor κB in Granulocyte‐Macrophage Colony‐Stimulating Factor Antagonism of Dexamethasone Suppression of the Macrophage Response toAspergillus fumigatusConidia

The dexamethasone (DEX) immunosuppressive effect on macrophage killing activity and cytokine production in response to Aspergillus fumigatus conidia is antagonized by granulocyte-macrophage colony-stimulating factor (GM-CSF). The molecular mechanism is unknown. We postulated that this antagonism is mediated by inhibitor kappaB (I kappaB) induction by DEX and is opposed by acceleration of I kappaB degradation by GM-CSF with or without conidia stimulation, with corresponding effects on translocation and activation of nuclear factor kappa B (NF-kappaB). We studied 2 types of cells, resident peritoneal macrophages from CD-1 mice and the murine macrophage RAW264.7 cell line. Cells were unstimulated or stimulated with conidia and simultaneously treated with DEX, GM-CSF, or DEX plus GM-CSF, for 2-4 hours. I kappaB degradation and NF-kappaB activation were assessed by Western blot. Macrophages stimulated with conidia alone increased NF-kappaB translocation. DEX increased I kappaB levels in cytoplasm and blocked translocation of NF-kappaB to the nucleus in unstimulated and conidia-stimulated macrophages. Conversely, GM-CSF decreased I kappaB levels. GM-CSF reversed the effect of DEX on I kappaB levels. NF-kappaB levels were minimal in DEX-treated macrophage nuclear extracts, compared with those from GM-CSF-treated and GM-CSF plus DEX-treated macrophages. GM-CSF can reverse the DEX immunosuppressive effect by enhancing I kappaB degradation and promoting NF-kappaB translocation. This would allow macrophage production of proinflammatory cytokines, facilitating resistance to aspergillosis.

Interaction between conidia, lung macrophages, immunosuppressants, proinflammatory cytokines and transcriptional regulation

The immunosuppressive effect of dexamethasone (DEX) on macrophage killing activity and cytokine production in response to Aspergillus fumigatus conidia is antagonized by granulocyte-macrophage colony-stimulating factor (GM-CSF). However, the intersection of signaling pathways and the molecular mechanism of this antagonism remain to be defined. We postulated that DEX inhibition of NF-kappaB was opposed by induction of IkappaB kinases (IKK) by GM-CSF + conidia stimulation, degradation of IkappaB, and release of nuclear factor kappa B (NF-kappaB). This hypothesis was tested using resident peritoneal macrophages from CD-1 mice and the murine macrophage RAW 264.7 cell line. Macrophages were unstimulated or stimulated with A. fumigatus conidia and simultaneously treated with DEX, GM-CSF or DEX + GM-CSF for 2 4 hours. IkappaB degradation in cytoplasmic extracts and translocation of NF-kappaB in nuclear extracts was measured by Western blot analysis. This showed GM-CSF reverses the immunosuppressive effect of DEX by enhancing the degradation of IkappaB and promoting the translocation of NF-kappaB to the nucleus. This would allow the production of proinflammatory cytokines by macrophages, facilitating resistance to A. fumigatus.

Impairment of Microbial Killing and Superoxide-Producing Activities of Alveolar Macrophages by a Low Level of Ozone.

Male Wistar rats were exposed to 0.2 ppm ozone for up to 14 days, during which alveolar macrophages were collected by pulmonary lavage to assess the effect of ozone on their microbial killing and superoxide-producing activities. For rapid assessment of microbial killing activity, we measured the release of 3H-radioactivity into the supernatant by deoxycholate-lysis of the macrophages that had phagosytosed and killed 3H-uridine-labeled microbes. The killing activity against Escherichia coli and Candida albicans was reduced to 70-80% of control levels on day 3. However, phagocytosis by and the activity of lysosomal enzymes of the macrophages were not impaired. On day 14 the killing activity against E. coli had returned to control levels, whereas that against C. albicans was still reduced. Because active oxygen species plays an important role in microbial killing activity of macrophages, the effects of ozone on respiratory burst and superoxide production were examined. Aliquots of alveolar macrophages were stimulated with phorbol myristate acetate (PMA), opsonized zymosan, or lipopolysaccharide (LPS) plus cytochalasin E (Cyt.E). The respiratory burst, oxygen consumption for rapid superoxide production, was decreased to 60-80% of control levels on day 3. On day 14, the respiratory burst by opsonized zymosan was still 80% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In addition, the superoxide-producing activity of ozone-exposed macrophages was 10-60% decreased on day 3. On day 14, the superoxide production by stimulation with opsonized zymosan was still 60% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In conclusion, because of their decreased production of superoxide, the host defense activity of alveolar macrophages was impaired by in vivo exposure to 0.2 ppm ozone. In particular, the C. albicans-associated defect lasted throughout the exposure period.

A new azole derivative of 1,4-benzothiazine increases the antifungal mechanisms of natural effector cells.

The most widely used drug for treatment of candidiasis is fluconazole (FCZ). Recently, a new derivative of 1,4-benzothiazine, compound FS5, was developed. FS5 had an appreciable protective effect against murine candidiasis. The present study was designed to dissect the antifungal mechanisms triggered by FS5 and to establish whether this compound could enhance the antimicrobial abilities of natural effector cells. The results show that intraperitoneal injection of FS5 in mice (i) induced an increase in circulating neutrophil levels comparable to that observed in FCZ-treated mice; (ii) enhanced phagocytosis and the killing activities of macrophages (Mphis) isolated from the spleen or peritoneal cavity, with the latter effect correlating with induction of nitric oxide synthesis and production by Mphis; and (iii) increased the levels of expression and synthesis of tumor necrosis factor alpha. These results suggest that the compound-induced synthesis of antimicrobial and proinflammatory molecules by heterogeneous Mphi populations is part of the beneficial effect of FS5 exerted against murine candidiasis.

Preferential Induction of TNF-α and IL-1β and Inhibition of IL-10 Secretion by Human Peripheral Blood Monocytes by Synthetic Aza–Alkyl Lysophospholipids

Several newly synthesized aza–alkyl lysophospholipids (AALP) have been shown to exert a potent antitumor cytotoxicityin vitro.Their potential usein vivoprompted us to study their effects on the immune system. The present study investigated the effect of AALP on the secretion of cytokines (TNF-α, IL-1β, IL-6, and IL-10) by normal and activated human peripheral blood-derived monocytes (PBM). Five AALP compounds (BN52205, BN52207, BN52211, BN52218, and BN52227) were tested. Human peripheral blood monocytes were cultured for 18 h at 37°C in the presence of AALP and/or LPS (1 μg/ml) or IFN-γ (1000 U/ml) and the supernatants tested for the presence of cytokines by ELISA. All five AALP compounds stimulated TNF-α and IL-1β secretion but not IL-6 secretion from nonstimulated PBM. There were no significant differences among the five AALP compounds tested and BN52207 was selected for further studies. Secretion of TNF-α was significantly potentiated by BN52207 when the PBM were activated by either IFN-γ or LPS. There was also an upregulation of TNF-α mRNA transcription as detected by RT-PCR. The induction of TNF-α secretion by BN52207 was dependent onde novoprotein synthesis as the specific TNF-α inhibitor, pentoxifylline, and the protein synthesis inhibitors, cyclohexamide and emetine, abolished TNF-α secretion. BN52207 also stimulated IL-1β secretion by resting and activated PBM in a concentration-dependent manner. Unlike TNF-α and IL-1β, however, BN52207 had no effect on IL-6 secretion. Noteworthy, unlike the induction of TNF-α and IL-1β secretion, BN52207 inhibited completely the secretion of IL-10 by resting and LPS-activated PBM. Further, BN52207 enhanced the macrophage killing activity of tumor target cells. Overall, this study demonstrates that AALP are endowed with a selective regulation of cytokine synthesis and secretion by resting and activated PBM. This regulation is manifested by upregulating TNF-α and IL-1β secretion and abolishing IL-10 secretion. The selective regulation of cytokine synthesis and secretion by AALP suggest that AALP may have potential therapeutic usesin vivoin clinical disease manifestations that are regulated by cytokines.

Role of interleukin-2 receptor expression on macrophages from Salmonella-infected mice

In this study, it was found that macrophages from Salmonella-infected mice expressed the interleukin-2 receptor (IL-2R). The IL-2R induced by the infection consisted mainly of both β and γ chains, and the expression of IL-2Rα was very low. Furthermore, similar expression of IL-2Rβ and γ chains was observed in interferon-γ-treated macrophages. The effect of recombinant interleukin-2 on the intracellular killing activity of macrophages which expressed IL-2Rβ and γ was then studied. Our results demonstrated that interleukin-2 acted synergistically with interferon-γ through the IL-2R in the induction of macrophage intracellular killing activity and established a new role of IL-2R on bactericidal activity of macrophages.

Role of interleukin-2 receptor expression on macrophages from Salmonella-infected mice.

In this study, it was found that macrophages from Salmonella-infected mice expressed the interleukin-2 receptor (IL-2R). The IL-2R induced by the infection consisted mainly of both beta and gamma chains, and the expression of IL-2Ralpha was very low. Furthermore, similar expression of IL-2Rbeta and gamma chains was observed in interferon-gamma-treated macrophages. The effect of recombinant interleukin-2 on the intracellular killing activity of macrophages which expressed IL-2Rbeta and gamma was then studied. Our results demonstrated that interleukin-2 acted synergistically with interferon-gamma through the IL-2R in the induction of macrophage intracellular killing activity and established a new role of IL-2R on bactericidal activity of macrophages.

Selective induction of interleukin-1 production and tumor killing activity of macrophages through apoptosis by the inhibition of oxidative respiration.

Suppression of mitochondrial respiration and increased glycolysis are characteristic features of activated macrophages. We show here that antimycin A, a respiratory inhibitor, induced interleukin-1 synthesis and tumoricidal activity without inducing tumor necrosis factor or nitric oxide. The induction of tumoricidal activity was resistant to inhibitors of tyrosine-specific protein kinases and intracellular glycoprotein transport. The cognate interaction between macrophages and target cells was not a prerequisite for the tumoricidal activity. In contrast, lipopolysaccharide induced the production of interleukin-1, tumor necrosis factor and nitric oxide, the induction of tumoricidal activity being sensitive to genistein and brefeldin A. Antimycin A, like lipopolysaccharide, induced the release of a cytoplasmic enzyme and apoptosis of macrophages. Antimycin A showed anti-metastatic activity in vivo. These results suggest that the inhibition of oxidative respiration would induce apoptosis and the resultant release of soluble effector molecules of macrophages which inhibit tumor metastasis in vivo.

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4+ and CD8+ T cells and an increase of activated T cells expressing IL-2R alpha were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-gamma was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-gamma in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-gamma was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-gamma plays an important role in the protection of mice from P. carinii infection.

Effects of sub-minimal inhibitory concentrations of antimicrobial agents on the cell surface of Klebsiella pneumoniae and phagocytic killing activity.

Changes in the phagocytic killing activity, capsule structure, and physicochemical properties such as the hydrophobicity and charge of the cell surface were studied in Klebsiella pneumoniae treated with sub-minimal inhibitory concentrations (MICs) of various antimicrobial agents. The phagocytic killing activity of macrophages was enhanced by penicillins, cephems, and monobactam in the absence of antibodies specific to the capsule or complement. No enhancement was observed with new quinolones, aminoglycosides, macrolide, or carbapenem. The thickness of the capsule structure was considerably reduced after the treatment with penicillins, cephems, and monobactam compared with the untreated control, and it was slightly reduced by new quinolones. No changes were observed in the capsule structure with aminoglycosides, macrolide, and carbapenem. The hydrophobicity on the cell surface of the bacteria was considerably increased after the treatment with penicillins, cephems, and monobactam compared with the control, slightly increased with new quinolones and carbapenem, and not changed with aminoglycosides and macrolide. The negative charge of the cell surface of the bacteria was reduced by penicillins, cephems, and monobactam compared with the control. It was slightly reduced by new quinolones and carbapenem but was not reduced by aminoglycosides and macrolide. These findings suggest that sub-MIC beta-lactam drugs such as penicillins, cephems, and monobactams cause thinning of the capsule of K. pneumoniae with increases in the hydrophobicity and decreases in the negative charge of the cell surface, which reduces the physical repulsion between the K. pneumoniae and phagocytes and enhances the sensitivity of the bacteria to phagocytic killing activity.

A therapeutic trial of experimental tuberculosis with gamma-interferon in an immunocompromised mouse model

It has been well documented that IFN-gamma plays an important role in the host defense against Mycobacterium tuberculosis infection through activating macrophages to kill the organism. In the present study, we studied the effects of in vivo injection of monoclonal antibody against IFN-gamma (anti-IFN-gamma mAb) on the mycobacterial infection to confirm the role of this cytokine. The injection of anti-IFN-gamma mAb suppressed the enhanced expression of MHC class II and ICAM-1 on pulmonary parenchymal macrophages induced by intravenous injection of Mycobacterium bovis BCG. The number of bacilli recovered from lung of mice treated with anti-IFN-gamma mAb and injected with Mycobacterium tuberculosis H37Rv was significantly larger than that of the control infected mice. From these results, it was indicated that anti-IFN-gamma mAb blocked the activities of endogenously synthesized IFN-gamma, thus inhibited the activation of macrophages to kill the bacilli. Next, CD4+ T cell-depleted mice were prepared by injecting anti-CD4 mAb and used as immunocompromised animal. When infected with M. tuberculosis, the multiplication of the bacilli within the lungs of such immunocompromised mice was much more enhanced in comparison with the control mice with intact CD4+ T cells. Administration of IFN-gamma significantly reduced the number of the bacilli in lung. Further, in an in vitro study with human lung macrophages, IFN-gamma enhanced the killing activity of macrophages against M. tuberculosis in a dose dependent manner, and suboptimal dose of 1 alpha, 25-dihydroxyvitamin D3 synergistically augmented the effect of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the murine interleukin-4 gene in an attenuated aroA strain of Salmonella typhimurium: persistence and immune response in BALB/c mice and susceptibility to macrophage killing

Cytokines are potentially useful in vaccination as adjuvants or modulators of the type of response induced. The work below describes the expression of a cloned cytokine gene for murine interleukin-4 (mIL-4) by a live vaccine vector, an attenuated aroA strain (SL7207) of Salmonella typhimurium, in a murine model system. SL7207 was used as a carrier for two different high-level expression vectors. Both resulting strains, designated SL7207(pOmpAmIL-4) and SL7207(pKKmIL-4), expressed the cloned gene product as monitored by both immunological and biological assays. However, SL7207(pOmpAmIL-4) produced mIL-4 at higher levels and was more stable in vitro than SL7207(pKKmIL-4). When SL7207(pOmpAmIL-4) was used as a live vaccine in BALB/c mice, this strain grew and survived at higher levels than the parental attenuated strain or empty plasmid-carrying strain in spleens, livers, and intestines. This difference in growth and survival did not appear to be caused by alterations in specific lymphocyte-mediated anti-Salmonella immune responses such as delayed-type hypersensitivity or serum antibody as measured by enzyme-linked immunosorbent assay; such alterations have been induced by IL-4 administration in other in vivo systems, and the lack of effect here may reflect the fact that IL-4 is not secreted from the bacteria in large quantities, most of the cytokine being in the cytoplasmic-membrane-bound fraction. Conversely, the ability of mouse macrophages to kill the bacteria in vitro was inhibited by bacterial production of mIL-4. This reduction in macrophage killing activity suggests that bacterial production of mIL-4 may be detrimental to host defense against Salmonella infection and may explain the enhanced bacterial growth and survival in vivo.

Genetic control of macrophage susceptibility to infection by Legionella pneumophila.

Legionella pneumophila readily grows in cultures of thioglycollate (TGC)-induced macrophages (MPs) from A/J mice, but not in MPs from BALB/c mice or other mouse strains. In the present study, the growth of Legionella pneumophilia in MPs from A/J and BALB/c mice, as well as hybrids of the two strains and back-crossed mice, was investigated to determine whether the permissiveness of growth of these bacteria was due to an inherited trait of the MPs. The MPs from all A/J mice supported the growth of Legionella, regardless of whether they were obtained from TGC or casein injected donors, but the cells from the mice given TGC supported growth of L. pneumophila much better than cells from mice injected with casein. Furthermore, MPs obtained from all BALB/c mice treated with either TGC or casein were nonpermissive for the growth of L. pneumophila. MPs from approximately 46% of the back-crossed ACF1 to A/J mice were permissive for L. pneumophila growth, while MPs from all ACF1 to back-crossed BALB/c mice were found to be nonpermissive. MPs from approximately 19% of ACF2 mice were permissive for L. pneumophila. Killing activities of MPs using temperature sensitive mutants of Salmonella typhimurium were variable and did not correlate with permissiveness or nonpermissiveness for growth of L. pneumophila.(ABSTRACT TRUNCATED AT 250 WORDS)