Immune suppression and induction of gamma interferon by pertussis toxin.

It has been suggested that pertussis toxin is a virulence factor of Bordetella pertussis. Although extracts enriched in pertussis toxin activity have been reported to enhance immune responsiveness, other studies have demonstrated a suppressive ability, suggesting that the toxin may contribute to the virulence of B. pertussis through mechanisms involving immune suppression. We report that purified pertussis toxin suppressed the in vitro immunoglobulin M antibody response of mouse splenocytes to sheep erythrocytes. At submitogenic doses, the toxin also suppressed [3H]thymidine incorporation by splenocytes, suggesting that it interfered with antibody formation by inhibiting lymphocyte proliferation. Antiviral activity was detected in culture supernatants obtained from pertussis toxin-suppressed splenocyte cultures by using a cytopathic effect inhibition assay. This antiviral activity was virus nonspecific, sensitive to pH 2.0 treatment, stable to heating at 56 degrees C, and neutralized by anti-gamma interferon antiserum. Finally, the fractionation of splenocytes by anti-immunoglobulin panning techniques suggested that Lyt2+ lymphocytes proliferated in response to pertussis toxin and produced interferon. Our results suggest that pertussis toxin may contribute to the virulence of B. pertussis through stimulation of Lyt2+ lymphocytes, resulting in the induction of gamma interferon and the subsequent inhibition of the primary antibody response.     

A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock.

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.     

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.     

High-level induction of gamma interferon with various mitogens in mice pretreated with Propionibacterium acnes.

Various T-cell mitogens induced high levels of circulating gamma interferon (IFN-gamma) in mice that had been pretreated with Propionibacterium acnes. Administration of lipopolysaccharide, a B-cell mitogen, to these mice also caused pronounced production of IFN-gamma in addition to IFN-alpha and IFN-beta. The enhanced induction was most marked at about 1 week after the pretreatment.     

Interleukin 2 enhances natural killer cell activity through induction of gamma interferon

Highly purified interleukin 2 (IL 2), free of interferon activity, enhanced natural killer (NK) cell activity against tumor cells in mouse spleen cell cultures and in human peripheral lymphocyte cultures in a manner similar to that of interferon (IFN). We determined that IL 2 enhanced NK activity indirectly in a cascade manner by the induction of gamma IFN (IFN-gamma) in the cultures, which actually mediated the enhanced killing. Accordingly, lymphocyte cultures treated with IL 2 alone produced 10 to 100 U of IFN per ml in 6 to 24 h of culture. The IFN was typed as IFN-gamma by specific antibodies. Specific antibodies either to natural IFN-gamma or to a synthetic peptide corresponding to the human IFN-gamma N-terminal amino acids, when added to cultures treated with IL 2, completely blocked IL 2 enhancement of NK cell activity for both the mouse and human systems. IL 2-induced proliferation was not affected by the antibodies. Thus, the enhancement of NK cell activity by IL 2 is completely mediated by IL 2-induced IFN-gamma. The findings clearly indicate a cascade effect whereby one lymphokine (IL 2) induces the production of another. The latter lymphokine (IFN-gamma) then mediates an important biological effect (natural killing).     

Experimental infection of neonatal foals with Rhodococcus equi triggers adult-like gamma interferon induction

Rhodococcus equi is a facultative intracellular pathogen that causes pneumonia in young foals but does not induce disease in immunocompetent adult horses. Clearance of R. equi depends mainly on gamma interferon (IFN-gamma) production by T lymphocytes, whereas the predominance of interleukin 4 (IL-4) is detrimental. Young foals, like neonates of many other species, are generally deficient in the ability to produce IFN-gamma. The objective of this study was to compare the cytokine profiles, as well as cell-mediated and antibody responses, of young foals to those of adult horses following intrabronchial challenge with R. equi. The lymphoproliferative responses of bronchial lymph node (BLN) cells to concanavalin A were significantly higher in foals than in adult horses. In contrast, adult horses had significantly higher lymphoproliferative responses to R. equi antigens than did foals. Infected foals had significantly lower IL-4 mRNA expression but significantly higher IFN-gamma expression and IFN-gamma/IL-4 ratio in R. equi-stimulated BLN lymphocytes than did infected adults. Infection with R. equi in foals resulted in a significant increase in the percentage of T lymphocytes and CD4(+) T lymphocytes in bronchoalveolar lavage fluid in association with a significant decrease in the percentage of these cell populations in BLNs. Infection of foals also resulted in a marked increase in serum immunoglobulin Ga (IgGa) and IgGb levels, resulting in concentrations in serum that were significantly higher than those of adult horses. This study demonstrates that the immune response to R. equi in foals is not biased toward IL-4 and is characterized by the predominant induction of IFN-gamma.     

Role of gamma interferon in Helicobacter pylori induction of inflammatory mediators during murine infection

Gamma interferon (IFN-gamma) has been proposed to play an important role in Helicobacter-related gastritis. Using the IFN-gamma gene knockout (IFN-gamma(-/-)) mouse model and a murine gastric epithelial cell line, GSM06, we demonstrated that Helicobacter pylori maximally induced macrophage inflammatory protein-2 (MIP-2) and inducible nitric oxide synthase (iNOS) mRNA only in wild-type mice. MIP-2 and iNOS mRNA were also induced by H. pylori in GSM06 cells. Induction of cyclooxygenase 2 mRNA through IFN-gamma was demonstrated in GSM06 cells. These data indicate that IFN-gamma mediates the induction of MIP-2 and iNOS mRNA expression by H. pylori in mice.     

Recombinant human gamma interferon inhibits simian malaria.

Prophylactic treatment with 0.1 mg of human gamma interferon per kg (body weight) per day completely suppressed experimental infection with Plasmodium cynomolgi B sporozoites in rhesus monkeys. Treatment with lower doses partially suppressed this infection. Prophylactic treatment with human gamma interferon, however, had no protective effect against trophozoite-induced infection, suggesting that the interferon effect was limited to the exoerythrocytic stage of parasitic development.     

Capacity of recombinant gamma interferon to activate macrophages for Salmonella-killing activity.

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.     

Immune-specific gamma interferon production correlates with lymphocyte blastogenesis.

Gamma interferon (IFN-gamma) production, measured by a new commercially available radioimmunoassay, and lymphocyte blastogenesis were investigated in human peripheral lymphocyte cultures from healthy adults stimulated by crude cytomegalovirus (CMV) antigen. Mononuclear cells were obtained from 32 healthy adults (18 CMV seropositive [S+] and 14 CMV seronegative [S-]) by Ficoll-Hypaque gradients and cultured in microtiter plates containing CMV antigen. Lymphocyte blastogenesis ([3H]thymidine uptake) and IFN-gamma were determined on day 6. The mean stimulation index for S+ individuals was significantly greater than that for S- individuals (P less than 0.001). Similarly, the IFN-gamma stimulation index was greater for S+ individuals than for S- individuals (P less than 0.005). A significant increase in the concentration of IFN-gamma (10 NIH units/ml) was observed for S+ individuals at 24 h of antigen stimulation, with peak levels at 4 days. The radioimmunoassay for IFN-gamma production by antigen-stimulated lymphocytes in vitro (IMRX; Centocor Inc., Malvern, Pa.) is a rapid and sensitive measure of cell-mediated immunity.     

Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.

Candidacidal activity in macrophages activated by recombinant gamma interferon was examined kinetically in relation to acidification of phagolysosomes. In resident peritoneal macrophages (PMPs) of BALB/c mice, enhanced killing activity against Candida albicans was demonstrated after incubation with 100 U of gamma interferon per ml for 24 h but not after incubation for 48 to 72 h. Conversely, increased generation of H2O2 was exhibited in PMPs incubated from 48 to 72 h but not in PMPs incubated for 24 h. In normal PMPs, fusion of lysosomes to candida-containing phagosomes was readily accomplished and phagosome-lysosome fusion was not enhanced further by activation. The candidacidal substance was extracted from granule-rich fractions of either normal or activated PMPs by using citric acid (pH 2.7) in equal amounts; the substance showed a noncationic, heat-stable protein nature. In addition, when phagolysosomal pH was determined by flow cytometry of intraphagolysosomal fluorescein isothiocyanate-labeled C. albicans, phagolysosomes with low pH (less than 4.0) were detected in about 40% of PMPs activated for 24 h but not in those activated for 72 h or in normal PMPs. Moreover, increasing the intralysosomal pH with NH4Cl resulted in a significant reduction of candidacidal activity in activated PMPs. These results indicate that the candidacidal activity of gamma interferon-activated PMPs correlates well with enhanced acidification of their phagolysosomes and suggest that the candidacidal activity of activated PMPs is independent from reactive oxygen molecules and is mediated by proteinaceous substance(s) generated only in a strong acidic milieu of phagolysosomes by activation.     

Endotoxin-induced serum factor that stimulates gamma interferon production.

Serum from Mycobacterium bovis BCG-infected mice that had been challenged with lipopolysaccharide (LPS) exhibited a marked ability to induce gamma interferon (IFN-gamma) in cultures of spleen cells of normal mice in the presence of interleukin-2 (IL-2). The inducing activity became detectable in the circulatory system about 90 min after LPS challenge, disappeared at around 5 h, and was observable upon 640x dilution of the serum. Addition of monoclonal anti-IL-2 receptor antibody to the culture inhibited the induction by the serum. The activity induced high levels of IFN-gamma even in nylon wool-nonadherent cells, while concanavalin A failed to do so. Serum from uninfected mice challenged with LPS contained no such activity. The molecular weight of the active substance, estimated by gel filtration, was about 70,000. There were pronounced differences among mouse strains in the activities of the sera prepared, which paralleled the amounts of IFN-gamma produced in vivo. However, the levels of IFN-gamma produced in whole spleen cells and in nylon wool-nonadherent cells from mice of various strains were the same when stimulated with competent serum. These results indicate the existence of an unidentified factor that induces IFN-gamma in cooperation with IL-2 in macrophage-depleted splenocytes. They also suggest that IFN-gamma production in vivo is not genetically controlled at the lymphocyte level but, rather, at the level of synthesis of the unknown factor.     

Role of gamma interferon in the pathogenesis of bacteremic pneumococcal pneumonia.

Gamma interferon (IFN-gamma) concentrations in blood, but not in lungs, rose significantly at 24 to 48 h after murine pulmonary infection with virulent pneumococci. In contrast, infection with avirulent pneumococcal strains produced minimal rises in serum IFN-gamma concentrations. Compared with that of immunocompetent mice, mortality was appreciably increased after pulmonary infection of IFN-gamma gene knockout mice, suggesting a protective role for IFN-gamma in host response to pneumococcal disease.     

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.     

In vitro gamma interferon tests for the detection of tuberculosis infection

The QuantiFERON-TB Gold (QFT-G) is a new FDA approved test for diagnosing Mycobacterium tuberculosis infection. This test detects the release of interferon-gamma (IFNgamma) in whole blood from sensitized persons when it is incubated with mixtures of synthetic peptides representing 2 proteins present in M. tuberculosis: early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10). These antigens have greater specificity than tests using purified protein derivative (PPD) as the tuberculosis antigen. The CDC recommends that QFT-G may be used in all circumstances in which the TB skin test is currently used, including contact investigations, evaluation of recent immigrants, and sequential-testing surveillance programs for infection control (e.g., those for health-care workers). Advantages of the QFT-G include: no false-positive reactions from previous BCG vaccination; no cross-reactivity with non-tuberculous mycobacterium infections; and, better detection of active tuberculosis.     

Simultaneous induction of interferon gamma and tumor necrosis factor alpha by different seleno-organic compounds in human peripheral blood leukocytes.

Ebselen is known as anti-inflammatory and anti-oxidant selenium containing drug. We have synthetized 13 seleno-organic compounds, analogs of ebselen. Seven of them were found to be inducers of interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) in human peripheral blood leukocytes (PBL) cultures. The most active cytokine inducers were: 2-phenyl-1,2-benzisoselenazol-3(2H)-one (1, ebselen), bis [2-(N-phenylcarbamoyl)]phenyl diselenide (7) and bis (2-[N-(2-pyridyl)carbamoyl])phenyl diselenide (8). The amounts of IFN and TNF produced by PBL cultures in response to the seleno-organic compounds were found to be similar to that induced by phytohemagglutinin (PHA). The activities of the seleno-organic compounds were dose-dependent and related to the chemical structure of the drugs suggesting involvement of the specific cytokine-inducer receptor. The simultaneous inductions of IFN-gamma and TNF-alpha were highly correlated, but independent on each other.     

Variation of interferon induction at the bone marrow level. Studies on interferon induction in relation to natural cell-mediated cytotoxic mechanisms

The effects of interferon inducers on different cytolytic mechanisms were studied in the high leukemia mouse strain AKR. A clear depression in baseline cytolytic potential and interferon-mediated stimulation of natural killer cell activities was demonstrated. This depression was most pronounced after 8 weeks of age. In contrast, antibody-dependent, cell-mediated cytotoxicity against IgG-coated chicken red blood cells was always normal. Bone marrow chimeras between CBA and AKR mice were produced to investigate the influence of bone marrow vs. host-mediated factors in these two strains with regard to interferon induction and cytolytic functions. Bone marrow genotype was found to be the dominating factor with regard to both parameters. Mice reconstituted with AKR bone marrow were deficient both in interferon production using tilorone and Newcastle disease virus as inducers, and at the level of natural killer cells responding to exogenously administered interferon. The possible relationship between these findings and the development of lymphomas in AKR mice is discussed.     

Fungicidal activation of murine macrophages by recombinant gamma interferon.

Alveolar macrophages from BALB/c mice readily phagocytized endospores (2 to 5 micron) and arthroconidia of Coccidioides immitis in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into spherules, and the arthroconidia formed germ tubes and hyphae. However, these processes did not occur if the macrophages were incubated with murine recombinant gamma interferon (rIFN-gamma) during infection with C. immitis. Treatment with rIFN-gamma activated the fungicidal capabilities of the alveolar macrophages, as evidenced by the 50% reduction in the CFU which could be recovered from macrophages infected in the presence of gamma interferon compared with alveolar macrophages infected without gamma interferon (P less than 0.05). Similar results were seen with peritoneal macrophages incubated with rIFN-gamma and infected with C. immitis. As little as 10 U of rIFN-gamma per ml reduced by half the number of C. immitis CFU which could be recovered from the phagocytes 8 h after infection with arthroconidia, although interferon alone did not affect the viability of the fungi.