Gamma interferon production in allogeneic stimulation: antigenicity that is sufficient to cause interferon production.

Spleen cells obtained from allogeneic cell-primed mice produced immune interferon when cocultivated with the antigenic cells. The purpose of this study was to clarify the antigenic determinants for immune interferon production in this allogeneic stimulation system. An incompatibility at the K end alone or at the D end alone of the H-2 complex was sufficient for immune interferon induction. No interferon production was observed in the combinations between strains of mice that differ for the non-H-2 regions, including the M locus. Immune interferon-producing cells (IIPC), induced by difference of the H-2K or H-2D regions, recognized the specificities controlled by the H-2K or H-2D regions, respectively; namely, there was no cross-reaction between the two regions. The difference between H-2d and H-2b did not cause interferon production in the combinations of which non-H-2 regions were very similar to each other. IIPC were not induced in the combination, but when IIPC were properly induced in B6 spleen cells, the IIPC could recognize the specificities controlled by the H-2d region (B10D2) and produced immune interferon.     

STAT1 is essential for antimicrobial effector function but dispensable for gamma interferon production during Toxoplasma gondii infection

The opportunistic protozoan Toxoplasma gondii is a prototypic Th1-inducing pathogen inducing strong gamma interferon (IFN-gamma) cytokine responses that are required to survive infection. Intracellular signaling intermediate STAT1 mediates many effects of IFN-gamma and is implicated in activation of T-bet, a master regulator of Th1 differentiation. Here, we show that T. gondii-infected STAT1-null mice fail to upregulate the IFN-gamma-dependent effector molecules inducible nitric oxide synthase (iNOS), IGTP, and LRG-47, which are required for mice to survive infection. Both T-bet and interleukin-12 receptor beta2 (IL-12Rbeta2) failed to undergo normal upregulation in response to T. gondii. Development of IFN-gamma-producing CD4(+) and CD8(+) T lymphocytes was severely curtailed in the absence of STAT1, but a substantial level of STAT1-independent non-T-cell-derived IFN-gamma was induced. Absence of STAT1 also resulted in increased IL-4, Arg1, Ym1, and Fizz1, markers of Th2 differentiation and alternative macrophage activation. Together, the results show that T. gondii induces STAT1-dependent T-lymphocyte and STAT1-independent non-T-cell IFN-gamma production, but that effector functions of this type 1 cytokine cannot operate in the absence of STAT1, resulting in extreme susceptibility to acute infection.     

Gamma interferon is essential for clearing Mycobacterium genavense infection

Factors determining the in vivo replication of the opportunistic pathogen Mycobacterium genavense are largely unknown. Following intravenous injection of a patient isolate, M. genavense could not be recovered by culture or detected by PCR in the livers or spleens of infected BALB/c mice. In contrast, M. genavense was found to chronically persist and multiply in the livers and spleens of intravenously infected syngeneic gamma-interferon-gene-deficient (GKO) mice as evidenced by acid-fast stains of infected tissues and recovery by both PCR and liquid culture following organ homogenization. In GKO mice, M. genavense elicited a chronic inflammatory response, resulting in marked splenomegaly and extensive lymphadenopathy. Granulomatous lesions in the livers of GKO mice were diffuse, were composed of monocytes, neutrophils, and CD3(+) cells, and were histochemically negative for inducible nitric oxide synthase.     

Gamma interferon inhibits production of Anti-OspA borreliacidal antibody in vitro

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-gamma). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-gamma was not affected by the time of treatment. In addition, treatment with rIFN-gamma inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-gamma marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-gamma does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.     

T cell-derived IL-3 plays key role in parasite infection-induced basophil production but is dispensable for in vivo basophil survival

Enhanced basophil production is often associated with T(h)2-related conditions such as parasite infections or allergic inflammations. Our previous study demonstrated that T cell activation is necessary to promote basophil production in Nippostrongylus brasiliensis (Nb)-infected mice. Yet, mechanisms underlying how T cells aid infection-induced basophil production are not clear. In this report, we show that IL-3 produced by T cells activated by the infection enhances basophil production in Nb-infected mice. IL-3-deficient mice or Rag2-/- recipients of IL-3-deficient T cells but not of wild-type T cells failed to support basophil production following the Nb infection. Interestingly, although IL-3 was critical for preventing basophil apoptosis in vitro, IL-3 had little contribution to basophil survival and proliferation in vivo. Collectively, these results highlight a novel mechanism by which activation of adaptive immune components induces basophil production but not basophil survival via IL-3 production.     

Daily variation in macrophage phagocytosis is clock-independent and dispensable for cytokine production

Innate immune responses vary in a circadian manner, and more recent investigations aim to understand the underlying molecular mechanisms. Cytokine production varies significantly over the course of a day depending on the time of stimulation by pathogens or Toll-like receptor ligands, and multiple signaling pathways linked to the cell-autonomous circadian clock modulate innate immunity. Recognition of foreign material, especially by innate immune cells, engages a myriad of receptors, which trigger inflammatory responses, as well as endocytosis and degradation and/or processing for antigen presentation. Because of the close connection between particle engulfment and inflammation, it has been proposed that phagocytic uptake may drive cytokine production in phagocytes. Here we show that bacterial particle ingestion by mouse peritoneal macrophages displays temporal variation, but is independent of the cell-intrinsic circadian clock in an ex vivo setting. Although cytokine production is dependent on phagocytosis, uptake capacity across 12 hr does not translate into 24-hr rhythms in cytokine production. In vivo, time-of-day variations in phagocytic capacity are not found, whereas a time of day and clock-dependent cytokine response is maintained. These data show that efficiency of bacterial phagocytosis and the 24-hr rhythmicity of cytokine production by macrophages are independent of one another and should be studied separately.     

Gamma interferon production in endotoxin-responder and -nonresponder mice during infection.

The production of gamma interferon (IFN-gamma) in response to infection and to a number of other agents was compared in Lpsn (C3H/HeN and C57BL/10ScSn) and Lpsd (C3H/HeJ and C57BL/10ScCr) mouse strains. Large differences in IFN-gamma production were observed between C57BL/10ScCr mice and the other mouse strains. With the exception of C57BL/10ScCr, all mouse strains, including C3H/HeJ, exhibited transient levels of IFN-gamma during infection with Salmonella typhimurium. Spleen cells of these mice, explanted on day 3 of infection, produced in vitro IFN-gamma spontaneously; this production was enhanced considerably by heat-killed S. typhimurium, heat-killed Propionibacterium acnes, concanavalin A (ConA), or lipopolysaccharide (LPS). These stimuli, except for LPS, also induced IFN-gamma production in cultures of normal spleen cells from noninfected animals. In contrast, C57BL/10ScCr mice produced no IFN-gamma following infection with S. typhimurium. Also, spleen cells of these mice, explanted on day 3 of infection, exhibited no spontaneous IFN-gamma production. A marginal response was obtained by additional stimulation of the cells with killed S. typhimurium, and a moderate response was obtained with ConA. Normal spleen cells from noninfected C57BL/10ScCr mice showed no IFN-gamma response to killed S. typhimurium, killed P. acnes, or LPS and only a low response to ConA. Impaired IFN-gamma production in C57BL/10ScCr mice was also evident during infection with Plasmodium chabaudi chabaudi, with which a low IFN-gamma response was seen only occasionally. Also, spleen cells from infected animals (days 2 to 8 after infection) exhibited only a very low level of IFN-gamma production in vitro; however, this production could be enhanced further by ConA. In comparison, C57BL/10ScSn mice infected with P. chabaudi chabaudi produced significant amounts of IFN-gamma. Spleen cells explanted from infected animals produced IFN-gamma spontaneously in vitro; this production was enhanced further by killed P. acnes and ConA. The results showed that in addition to the defect in LPS responsiveness, C57BL/10ScCr mice possess a defect in IFN-gamma production in response to different stimuli. During infection, IFN-gamma production and sensitization to LPS occurred in parallel. Infected Lpsn mice exhibited enhanced sensitivity and infected Lpsd C3H/HeJ mice exhibited reasonable sensitivity to the lethal effects of LPS. Lpsd C57BL/10ScCr mice remained resistant to LPS when infected with S. typhimurium and exhibited only marginal sensitivity when infected with P. chabaudi chabaudi.     

Inosiplex-dependent interferon production in vivo and in vitro

Inosiplex, an immunopotentiator, was shown to be capable of inducing interferon synthesis in vivo and in cell culture. Its concentrations of the order of 25 mg/mouse were to be most effective. The time course of production showed a late peak at 72 hours. The resulting interferon was not neutralized by antiserum to alpha- and beta-interferons. L929 cells of peritoneal exudate and splenocytes could synthesize interferon after treatment with inosiplex. The use of mixed cultures of these cells increased the effectiveness of interferon induction.     

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.     

Gamma interferon is not required for arthritis resistance in the murine Lyme disease model

Lyme arthritis is the most common complication following infection of human individuals with Borrelia burgdorferi sensu stricto. In mice, B. burgdorferi infection leads to arthritis of the tibiotarsal joints. Arthritis severity in mice is under host genetic control, as BALB/c mice developed mild arthritis but C3H/He mice developed severe disease following B. burgdorferi infection. To study the role of gamma interferon (IFN-gamma) in arthritogenesis, targeted mutant mice lacking the IFN-gamma receptor (IFN-gammaR) were infected by inoculation with B. burgdorferi. IFN-gammaR(-/-) and parental 129/SvEv mice developed mild arthritis of similar severity, as determined both by weekly tibiotarsal joint measurements and histopathology at 2 and 5 weeks postinfection. Both strains of mice had the same spirochetal burden in the joints, suggesting that the IFN-gammaR(-/-) mice were not impaired in controlling spirochetal expansion in vivo. The wild-type mice mounted a Th1 response, with a predominance of CD4(+) IFN-gamma(+) T cells observed by flow cytometry. In contrast, the IFN-gammaR(-/-) mice mounted a Th2 response, with a predominance of CD4(+) IL-4(+) T cells. As expected given their cytokine profile, the IFN-gammaR(-/-) mice produced fewer CD8(+) IFN-gamma(+) and MAC-1(+) IL-12(+) cells and less immunoglobulin G2a (IgG2a) than their wild-type counterparts. These results strongly suggest that IFN-gamma is not required for arthritis resistance or as part of an effective immune response against B. burgdorferi.     

Gamma interferon as a crucial host defense against Rickettsia conorii in vivo.

Gamma interferon (IFN-gamma) plays an important role as a host defense in rickettsial infection. Swiss Webster mice, which are resistant to Rickettsia conorii (Malish 7 strain) infection, were treated with a monoclonal antibody against mouse IFN-gamma. When the antibody-treated mice were inoculated with 12 50% tissue culture infective doses of R. conorii, the mortality was 47% and the morbidity was 100%. None of the control mice, which received the same dose of R. conorii, died or became ill. The enumeration of rickettsiae in organs by direct immunofluorescence in paraffin sections demonstrated higher quantities of rickettsiae in the spleen had liver of IFN-gamma-depleted mice as compared with those of the infected controls. The kinetic analysis of IFN-gamma levels in sera showed depletion in the treated mice. These results indicate that IFN-gamma plays an important role as a host defense in the early stage of rickettsial infection. Survival of some mice despite continued treatment with antibody to IFN-gamma suggests that other immune mechanisms may also be important.     

Gamma interferon is not essential in host defense against disseminated candidiasis in mice.

In vitro studies have suggested a role for interferon gamma (IFN-gamma) in host defense against disseminated candidiasis, but in vivo studies are inconclusive. We utilized homozygous IFN-gamma knockout (GKO) mice to determine if the cytokine is essential in host defense against this disease. Genotypes of mice were determined by PCR with specific primers for the normal or disrupted IFN-gamma gene. The GKO status of the mice was confirmed by an enzyme-linked immunosorbent assay, which showed no detectable IFN-gamma produced by their splenocytes stimulated by concanavalin A. To test the susceptibility of GKO mice to candidiasis, the animals were infected either intravenously (i.v.) or intragastrically (i.g.) with Candida albicans. GKO mice infected i.v. survived as long as wild-type (WT) mice and showed no difference in Candida CFU counts in liver, spleen, or kidneys compared to those for WT mice. When animals were given Candida i.g., at 3 h or at 10 or 21 days after infection, there was no dissemination of Candida to the lung, liver, spleen, or kidneys for either GKO or WT mice. There was no difference in Candida CFU counts recovered from the stomach or intestines between GKO and WT mice. Histological examination of the stomach cardial-atrium fold, where the fungus was located, showed that GKO mice did not have evidence of more tissue damage or fungal invasion than WT mice. Finally, the jejunum for both types of mice showed no evidence of tissue damage or fungal invasion. These studies indicate that IFN-gamma is not essential in host defense against C. albicans that originates from a mucosal site or that is given directly into the bloodstream in a mouse model.     

Gamma interferon treatment in vivo provokes accumulation of activated monocytes in the venous circulation of rats

Activated monocytes forming intravascular clumps in the veins of most organs appeared in LEW rats after a 3-day intravenous treatment with recombinant rat gamma interferon. Phenotyping in situ and in cytospot preparations of perfusates revealed that the cells coexpressed the rat monocyte/macrophage antigen ED1 and class II MHC molecules. In addition, most cells reacted with a rat CD11b antibody and weakly expressed determinants detected by the W3/13 and Ox22 reagents. Minor fractions of the activated monocytes were positive for rat CD4 and the Ox2 and ED3 determinants. Cell proliferation was assessed by double staining for bromodeoxyuridine (BrdUrd) incorporation and phenotypic markers. Of the ED 1-positive class II-positive cells, 80% were labeled with BrdUrd after 3 days of combined infusion with gamma interferon. Pulse labeling for 30 minutes revealed 8% BrdUrd-positive intravascular ED 1-positive class II-positive monocytes in situ on day 3 of treatment, which contrasted with almost-absent labeling of this cell population in normal LEW rats. It is concluded that interferon not only promotes activation but also intravascular division of monocytes or their immediate precursors. Interestingly, cells of identical morphology and phenotype were observed in the vasculature of rats during lethal graft-versus-host reactions. Activated monocytes may thus contribute to the pathologic consequences of cytokine treatment and severe systemic immune reactions in vivo.     

Gamma interferon treatment of patients with chronic granulomatous disease is associated with augmented production of nitric oxide by polymorphonuclear neutrophils

Treatment with gamma-interferon (IFN-gamma) is associated with reduced frequency and severity of infections in chronic granulomatous disease (CGD), but the mechanism is unknown. Since the inducible nitric oxide (NO) synthase can be amplified by IFN-gamma in murine macrophages, for example, we hypothesized that IFN-gamma might modulate NO release from polymorphonuclear neutrophils (PMNs) in patients with CGD. Eight patients with CGD and eight healthy controls were studied. Each patient was given either 50 or 100 microg of IFN-gamma per m2 on two consecutive days. The production of NO from N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs was assessed as the NG-monomethyl-L-arginine-inhibitable oxidation of oxyhemoglobin to methemoglobin in the presence of catalase and superoxide dismutase. Prior to IFN-gamma treatment, the PMNs from CGD patients produced 372 +/- 27 (mean +/- standard error of the mean) pmol of NO/10(6) PMNs at 45 min, while the control PMNs produced 343 +/- 44 pmol. On day 1 after IFN-gamma treatment, NO production increased to 132% +/- 25% of that for controls, and on day 3 it reached 360% +/- 37% (P < 0.001) of that for controls. On day 8, the values still remained higher, 280% +/- 78% more than the control values. Likewise, the bactericidal capacity of PMNs increased on day 3. The present data show that IFN-gamma treatment of CGD patients is associated with an increased production of NO from PMNs when activated by fMLP. Since these PMNs lack the capacity to produce superoxide anions, it is conceivable that this increase in NO release could be instrumental in augmenting host defense.     

The Mycobacterium tuberculosis ESAT-6 homologue in Listeria monocytogenes is dispensable for growth in vitro and in vivo

ESAT-6 is a virulence determinant in Mycobacterium tuberculosis and a member of a conserved group of proteins in a variety of other bacteria. A targeted deletion of the homologous gene in Listeria was generated, and in contrast to that observed for mycobacteria, this locus was not required for Listeria virulence.     

Cytokine induction of neopterin production.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.     

CD44 is dispensable for B lymphopoiesis

Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.     

Gamma interferon production by hepatic NK T cells during Escherichia coli infection is resistant to the inhibitory effects of oxidative stress

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-gamma) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-gamma in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-gamma-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-gamma-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1(+) CD3(-)), conventional T cells (NK1.1(-) CD3(+)), and NK T cells (NK1.1(+) CD3(+)). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-gamma production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-gamma expression by a subset of NK T cells, which did not bind alpha-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-gamma-expressing cells were also predominantly CD8(+) and bore gamma delta T-cell antigen receptors. The residual IFN-gamma response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.