Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages.

Lipopolysaccharide (LPS) derived from the periodontal pathogen Porphyromonas gingivalis has been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation of P. gingivalis LPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by this P. gingivalis LPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages by Escherichia coli LPS. These data suggest that (i) P. gingivalis LPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.

Impaired IFN-gamma production in IFN regulatory factor-1 knockout mice during endotoxemia is secondary to a loss of both IL-12 and IL-12 receptor expression.

Mice with a targeted mutation in the gene that encodes the transcription factor IFN regulatory factor-1 (IRF-1) were used to assess the contribution of IRF-1 to IL-12-dependent and IL-12-independent pathways of IFN-gamma production. In response to LPS, IRF. 1-/- mice produced less IL-12 p40, IL-12 p35, and IFN-gamma mRNA in the liver than IRF-1+/+ mice. While pulmonary IFN-gamma mRNA levels were also mitigated in IRF-1-/- mice, pulmonary IL-12 p40 and IL-12 p35 mRNA were not dysregulated. Circulating IL-12 p70 and IFN-gamma levels were profoundly attenuated in LPS-challenged IRF-1-/- mice. Further analysis revealed a major deficiency in hepatic IL-12Rbeta1 and IL-12Rbeta2 mRNA expression as well as pulmonary IL-12Rbeta1 mRNA expression in LPS-challenged IRF-1-/- mice. In vitro, IFN-gamma up-regulated IL-12Rbeta1 mRNA in macrophages from IRF-1+/+, but not IRF-1-/-, mice. IFN-gamma-induced IL-12Rbeta2 mRNA expression was also diminished in macrophages from IRF-1-/- mice. In contrast to IRF-1+/+ mice, administration of exogenous IL-12 to IRF-1-/- mice resulted in reduced serum IFN-gamma and hepatic and pulmonary IFN-gamma mRNA, demonstrating that loss of IL-12R results in diminished IL-12 responsiveness. While LPS-challenged IRF-1-/- mice also had reduced IL-15 mRNA levels, serum IL-18 responses were intact. Finally, induction of IRF-1 mRNA by LPS in livers of IFN-gamma knockout mice were markedly attenuated, suggesting a feedback amplification loop. These studies indicate that IRF-1 deficiency disrupts both IL-12-dependent and -independent pathways of IFN-gamma production and that IRF-1 is a critical transcription factor involved in the regulation of not only IL-12, but also IL-12R.

Interferon regulatory factor (IRF)-1 and IRF-2 regulate interferon gamma-dependent cyclooxygenase 2 expression.

Cyclooxygenases (Cox) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. Cox-2 is the inducible isoform that is upregulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. In this study, we demonstrate that interferon (IFN)-gamma alone or in synergy with lipopolysaccharide (LPS) or interleukin 1alpha induces Cox-2 expression in mouse peritoneal macrophages, which is paralleled by changes in Cox-2 protein levels and prostaglandin E(2) (PGE(2)) release. Induction of Cox-2 was abrogated in macrophages that lack IFN regulatory factor (IRF)-1, consistent with an attenuated hepatic mRNA response in IRF-1(-/-) mice injected with LPS. Conversely, the absence of IRF-2 in macrophages resulted in a significant increase in both basal and inducible Cox-2 gene and protein expression as well as IFN-gamma-stimulated PGE(2) release, identifying IRF-2 as negative regulator of this promoter. Two IFN stimulation response elements were identified in the mouse Cox-2 promoter that were highly conserved in the human Cox-2 gene. Both bind endogenous IRF-1 and IRF-2 and regulate transcription in an IRF-1/2-dependent manner. Our data demonstrate conclusively the importance of IFN-gamma as a direct activator and coactivator of the Cox-2 gene, and the central role of IRF-1/2 family members in this process.

IL-12 is dysregulated in macrophages from IRF-1 and IRF-2 knockout mice.

Macrophages derived from IFN-regulatory factor-1 (IRF-1) and IRF-2 knockout (-/-) and wild-type (+/+) mice were utilized to examine the role of these transcription factors in the regulation of IL-12 mRNA and protein expression. Induction of IL-12 p40 mRNA by LPS was markedly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages. In contrast, IRF-1(-/-), but not IRF-2(-/-), macrophages exhibited impaired LPS-induced IL-12 p35 mRNA expression. The ability of IFN-gamma to augment LPS-induced IL-12 p40 mRNA further when both stimuli were present simultaneously was significantly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages, with the most profound impairment observed for IRF-1(-/-) macrophages. Reductions in IL-12 mRNA expression after stimulation with LPS or LPS plus IFN-gamma were accompanied by substantial reductions in IL-12 p40 and IL-12 p70 protein in both IRF-1(-/-) and IRF-2(-/-) macrophages. Priming IRF-1(-/-) and IRF-2(-/-) macrophages with IFN-gamma for 24 h before LPS treatment partially restored impaired IL-12 mRNA and protein production in both IRF-1(-/-) and IRF-2(-/-) macrophages. Depressed IL-12 levels were paralleled by significant reductions in IFN-gamma mRNA expression in IRF-1(-/-) and IRF-2(-/-) macrophages. These results indicate that both IRF-1 and IRF-2 are critical transcription factors in the regulation of macrophage IL-12 and consequently IFN-gamma production.

Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo.

The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines. The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood. All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo. While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein. Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent. Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent. Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein. Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced. These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.

The transcription factor interferon regulatory factor 1 is expressed after cerebral ischemia and contributes to ischemic brain injury.

The transcription factor interferon regulatory factor 1 (IRF-1) is involved in the molecular mechanisms of inflammation and apoptosis, processes that contribute to ischemic brain injury. In this study, the induction of IRF-1 in response to cerebral ischemia and its role in ischemic brain injury were investigated. IRF-1 gene expression was markedly upregulated within 12 h of occlusion of the middle cerebral artery in C57BL/6 mice. The expression reached a peak 4 d after ischemia (6.0 +/- 1.8-fold; P < 0.001) and was restricted to the ischemic regions of the brain. The volume of ischemic injury was reduced by 23 +/- 3% in IRF-1(+/-) and by 46 +/- 9% in IRF-1(-/-) mice (P < 0.05). The reduction in infarct volume was paralleled by a substantial attenuation in neurological deficits. Thus, IRF-1 is the first nuclear transacting factor demonstrated to contribute directly to cerebral ischemic damage and may be a novel therapeutic target in ischemic stroke.

Induction of adrenomedullin mRNA and protein by lipopolysaccharide and paclitaxel (Taxol) in murine macrophages.

Lipopolysaccharide (LPS), a potent inflammatory stimulus derived from the outer membrane of gram-negative bacteria, has been implicated in septic shock. Plasma levels of adrenomedullin (AM), a potent vasorelaxant, are increased in septic shock and possibly contribute to the characteristic hypotension. As macrophages play a central role in the host response to LPS, we studied AM production by LPS-stimulated macrophages. When peritoneal exudate macrophages from C3H/OuJ mice were treated with protein-free LPS (100 ng/ml) or the LPS mimetic paclitaxel (Taxol; 35 microM), an approximately 10-fold increase in steady-state AM mRNA levels was observed, which peaked between 2 and 4 h. A three- to fourfold maximum increase in the levels of immunoreactive AM protein was detected after 6 to 8 h of stimulation. While LPS-hyporesponsive C3H/HeJ macrophages failed to respond to protein-free LPS with an increase in steady-state AM mRNA levels, increased levels were observed after stimulation of these cells with a protein-rich (butanol-extracted) LPS preparation. In addition, increased AM mRNA was observed following treatment of either C3H/OuJ or C3H/HeJ macrophages with soluble Toxoplasma gondii tachyzoite antigen or the synthetic flavone analog 5, 6-dimethylxanthenone-4-acetic acid. Gamma interferon also stimulated C3H/OuJ macrophages to express increased AM mRNA levels yet was inhibitory in the presence of LPS or paclitaxel. In vivo, mice challenged intraperitoneally with 25 microg of LPS exhibited increased AM mRNA levels in the lungs, liver, and spleen; the greatest increase (>50-fold) was observed in the liver and lungs. Thus, AM is produced, by murine macrophages, and furthermore, LPS induces AM mRNA in vivo in a number of tissues. These data support a possible role for AM in the pathophysiology of sepsis and septic shock.

Pulmonary and hepatic gene expression following cecal ligation and puncture: monophosphoryl lipid A prophylaxis attenuates sepsis-induced cytokine and chemokine expression and neutrophil infiltration.

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1beta, IL-6, gamma interferon (IFN-gamma), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-gamma-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-alpha, MCP-1, MIP-1alpha, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.

Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages.

Monophosphoryl lipid A (MPL) is a nontoxic derivative of the lipid A region of lipopolysaccharide (LPS) that is being developed as both an adjuvant and prophylactic drug for septic shock. We compared the ability of LPS and MPL to induce interleukin-10 (IL-10), IL-12 p35, IL-12 p40, gamma interferon (IFN-gamma), glucocorticoid receptor (GR), IL-1 receptor antagonist (IL-1ra), and inducible nitric oxide synthase mRNA expression in murine peritoneal macrophages. These genes were chosen for their ability to positively or negatively regulate the host immune response and thus for their potential involvement in MPL-induced adjuvanticity or in its ability to protect against sepsis. LPS was a more potent inducer of IL-12 p35, IL-12 p40, and IFN-gamma mRNA, as well as of IL-12 protein, than MPL. In contrast, MPL induced higher levels of IL-10 mRNA than did LPS from 1 to 1,000 ng/ml. In general, MPL was not a more potent inducer of negative regulatory genes, since MPL and LPS induced similar levels of GR and IL-1ra mRNA. Addition of anti-IL-10 antibody to cultures increased the induction of MPL-induced IL-12 p35, IL-12 p40, and IFN-gamma mRNA, suggesting that the enhanced production of IL-10 by MPL-stimulated macrophages contributes to decreased production of mRNA for IL-12 (p35 and p40) and IFN-gamma. Conversely, the addition of exogenous IL-10 to LPS-treated macrophages reduced the mRNA expression of these cytokine genes. These studies suggest that enhanced production of IL-10 by MPL-stimulated macrophages may contribute to the reduced toxicity of MPL through its negative action on induction of cytokines shown to enhance endotoxicity.

Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo: the roles of macrophages, endogenous IFN-gamma, and TNF receptor-1-mediated signaling.

To evaluate potential roles for macrophages, IFN-gamma, and TNF receptor 1 (TNFR1) in the regulation of LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, we used a model of macrophage depletion as well as IFN-gamma (GKO) and TNFR1 (TNFR1 -/-) knockout mice. LPS-induced iNOS mRNA in spleen was ablated in both macrophage-depleted and GKO mice. In livers of macrophage-depleted mice, LPS-induced iNOS mRNA was reduced by 55 to 85%, with the most profound reductions detected 6 and 8 h after LPS injection. In GKO mice, peak iNOS mRNA expression in liver (3 h) was unaffected by the loss of endogenous IFN-gamma. By 6 to 12 h after LPS challenge, however, hepatic LPS-induced iNOS mRNA and serum nitrate/nitrite levels were reduced substantially in GKO mice. Residual LPS-induced iNOS mRNA in livers of GKO mice was nearly ablated by macrophage depletion, indicating that induction of iNOS mRNA in liver requires both endogenous IFN-gamma and either macrophages and/or macrophage-derived factors. TNFR1-mediated signaling was involved in the induction of LPS-induced iNOS mRNA in liver at 3 and 6 h, but not in its maintenance at 8 h. Conversely, induction of iNOS mRNA in spleen by LPS was independent of TNFR1-mediated signaling. Our results indicate that macrophages and/or their secreted products, endogenous IFN-gamma production, and TNFR1-mediated signaling play key roles in the in vivo regulation of iNOS mRNA expression and that the extent of their involvement is both time and organ specific.

Differential dysregulation of nitric oxide production in macrophages with targeted disruptions in IFN regulatory factor-1 and -2 genes.

Recent studies have reported that IFN regulatory factor-1 (IRF-1) regulates nitric oxide (NO.) production in murine macrophages (M phi). Since IRF-2 recognizes the same consensus sequence as IRF-1, we postulated that IRF-2 may also regulate NO.. Therefore, the ability of M phi from INF-2 homozygous (IRF-2-/-) and heterozygous (IRF-2+/-) knockout mice to produce NO. following LPS and/or IFN-gamma stimulation was compared with the responses of IRF-1-/-, IRF-1+/-, and C57BL/6+/+ M phi. IRF-2-/- M phi produced less LPS-induced NO2- than IRF-2+/- or C57BL/6 M phi. LPS and IFN-gamma synergized to increase NO2- production from IRF-2-/- M phi to approximately 50% of IRF-2+/- and C57BL/6 levels. Unexpectedly, IRF-2-/-, IRF-2+/- and C57BL/6 M phi produced comparable levels of inducible NO synthase mRNA in response to treatment with LPS and IFN-gamma. IRF-1-/- M phi produced barely detectable NO2- and low, but detectable, inducible NO. synthase mRNA in response to IFN-gamma and LPS. These results indicate that IRF-1 and IRF-2 differ in their mechanism of NO. regulation and that IRF-2 regulates inducible NO. synthase post-transcriptionally.

Effect of liposome-mediated macrophage depletion on LPS-induced cytokine gene expression and radioprotection.

Tissue-specific cytokine mRNA expression was examined in mice that received LPS. In the liver, IL-6, IL-10, IL-12 (p40), and TNF-alpha were induced by 30 min after injection with LPS. In the spleen, IL-6 and TNF-alpha were induced by 30 min after LPS challenge, while increases in IL-10 and IL-12 (p40) were delayed in onset. GM-CSF, IFN-gamma, and IL-12 (p35) were not induced in the liver or spleen until 60 to 90 min after LPS injection. Mice were depleted of macrophages in their liver and spleen by i.v. injection of liposome-encapsulated dichloromethylene bisphosphonate (Cl2MBP). Induction of IL-1 beta, IL-6, IL-10, and IL-12 (p40) mRNA by LPS was reduced by > 95% in the liver of macrophage-depleted mice, implicating macrophages as the primary producers of these cytokines. Macrophage depletion resulted in a 50 to 75% reduction in TNF-alpha mRNA in the liver. The results from Cl2MBP-liposome-treated mice also suggested that splenic macrophages were the primary producers of LPS-induced IL-1 beta, IL-6, IL-12 (p40), and IL-1 receptor antagonist (IL-1ra) mRNA, but not IL-10 and TNF-alpha mRNA. Mice treated with Cl2MBP-liposomes were more susceptible to ionizing irradiation than control mice, whether or not they were administered a radioprotective dose of LPS. These findings suggest that depletion of liver and splenic macrophages results in a dysregulation of basal and LPS-induced cytokine responses that can be associated with an altered biologic response.

Differential expression of interferon regulatory factor 1 (IRF-1), IRF-2, and interferon consensus sequence binding protein genes in lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive macrophages.

Macrophages secrete interferon (IFN), as well as other cytokines, following lipopolysaccharide (LPS) stimulation. The interferon regulatory factors (IRFs) comprise a family of DNA-binding proteins that have been implicated in the transcriptional regulation of IFN and certain IFN-inducible genes. We therefore characterized basal and LPS-inducible levels of IRF-1, IRF-2, and interferon consensus sequence binding protein (ICSBP) mRNA in LPS-responsive macrophages and compared the expression of these genes in macrophages that typify two murine models of LPS hyporesponsiveness. In the first model, the LPS-hyporesponsive phenotype of the C3H/HeJ mouse is genetically determined and maps to the Lps locus on mouse chromosome 4. In the second model, normally LPS-responsive macrophages acquire a transient LPS-hyporesponsive phenotype following a prior exposure to LPS, a phenomenon referred to as "endotoxin tolerance." Using reverse transcription PCR, we detected basal levels of IRF-1 mRNA in LPS-responsive (Lpsn) macrophages that were approximately 15 times higher than those found in LPS-hyporesponsive (Lpsd) macrophages. Conversely, Lpsd macrophages expressed basal levels of IRF-2 mRNA that were approximately 18 times higher than those expressed in Lpsn macrophages. LPS stimulation resulted in a dose- and time-dependent accumulation of IRF-1, IRF-2, and ICSBP mRNA only in Lpsn macrophages. Cycloheximide inhibited the accumulation of LPS-stimulated IRF-2 and ICSBP mRNA, but not IRF-1 mRNA, thus designating IRF-1 an immediate-early, LPS-inducible gene. Finally, macrophages rendered tolerant to endotoxin expressed elevated but nonmaximal mRNA levels for all three transcription factors that are not reinduced upon secondary challenge with LPS. Thus, the IRFs may represent yet an additional molecular pathway in the complex response to LPS.

Taxol provides a second signal for murine macrophage tumoricidal activity.

The anticancer drug, taxol, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and bacterial LPS induce strikingly similar responses in murine macrophages. Here we report that taxol, like LPS, provides a "second" signal for murine macrophage activation to tumoricidal activity. Tumoricidal activity was determined by the release of 51Cr from prelabeled P815 mastocytoma target cells. Taxol or LPS alone weakly induced C3H/OuJ (Lpsn) murine macrophages to kill P815 mastocytoma cells, and tumoricidal activity was not induced by the classic "priming" signal, IFN-gamma. However, combinations of taxol or LPS with IFN-gamma synergized to activate macrophages to lyse tumor cells. Taxol activation of macrophages required an intact LPS signaling pathway, as taxol did not induce IFN-gamma-treated C3H/HeJ (Lpsd) macrophages to lyse target cells. In normal (Lpsn) murine macrophages, IFN-gamma, LPS, or taxol alone induced low or moderate levels of nitric oxide synthase gene expression and nitric oxide secretion. However, this gene and cytostatic metabolite were induced synergistically by combinations of taxol or LPS with IFN-gamma. Secretion of nitric oxide correlated with tumor cell killing, and taxol-activated macrophages failed to kill tumor targets in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase. The data illustrate the potential for taxol to activate macrophage mediated-antitumor mechanisms in addition to its better characterized role as an anti-mitotic agent.

Lipopolysaccharide increases glucocorticoid receptor expression in murine macrophages. A possible mechanism for glucocorticoid-mediated suppression of endotoxicity.

In a previous study we demonstrated that IFN-gamma induced an increase in the number of glucocorticoid receptors (GR) in murine macrophages. To examine further the environmental signals involved in regulation of macrophage GR availability, we asked whether another classical macrophage-activating factor, LPS, would induce an increase in GR number in the macrophage cell line, RAW 264.7, and in primary macrophages from C3H mice. We report that treatment of RAW 264.7 cells and peritoneal exudate macrophages from C3H/OuJ mice with protein-free, phenol water-extracted LPS (PW-LPS) induced an increase in the number of GR. A significant increase in GR number was observed as early as 4 h after PW-LPS treatment, was maximal at 12 h, and remained heightened through 48 h. Optimal induction of the GR by PW-LPS was observed when murine macrophages were treated with 10 ng/ml of PW-LPS. The LPS-induced increase in macrophage GR number could be inhibited by polymyxin B. Macrophages obtained from the LPS hyporesponsive C3H/HeJ strain did not respond to PW-LPS, but did respond to protein-rich, butanol-extracted LPS with a modest increase in GR number after treatment with 2 micrograms/ml. Moreover, taxol, an antineoplastic agent with LPS mimetic activity, also increased GR number in murine macrophages. These results suggest that LPS is not only an important macrophage-activating signal, but may also be important in sensitizing the cell for negative regulatory events such as feedback inhibition by glucocorticoids.

IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.

Activation of NK cells in mice following corneal infection with herpes simplex virus type-1.

Natural killer (NK) cells are large granular lymphocytes that mediate antigen nonspecific, non-major histocompatibility complex (MHC) restricted lysis of virus infected cells. They are thought to play a role in innate resistance to herpes simplex virus (HSV) infections. In most animal studies reported to date, the virus was injected intraperitoneally, not a natural route of infection. Using a murine model of acute HSV-1 ocular infection, we demonstrate that increased splenic NK activity is induced in BALB/c mice following corneal infection with four different strains of HSV-1. The kinetics of NK cell activation depended on the strain of virus and was associated with virulence of the strain and with the ability of the viruses to grow in vivo. We also assessed the role of interferon-alpha/beta, IFN-gamma, and interleukin-2 (IL-2) in the HSV-1 induced NK cell activation by treating mice with antisera against these lymphokines prior to infection. Treatment with anti-IFN-alpha/beta or anti-IFN-gamma significantly reduced NK cell cytotoxicity, suggesting that these lymphokines were involved in the activation of NK cells following HSV-1 ocular infection. Treatment with anti-IL-2 resulted in increased NK cell activity, suggesting that in vivo, IL-2 is involved in the suppression of NK cell activity in infected mice. Treatment with a combination of anti-IL-2 and anti-IFN also increased NK cytotoxicity. Despite the induction of high levels of NK activity, mice developed severe ocular disease or died of encephalitis.

Susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans.

The susceptibility of congenitally immunodeficient mice to a nonencapsulated strain of Cryptococcus neoformans (strain M7) was evaluated. Gnotobiotic mice with defined congenital defects in innate immunity (beige) or cell-mediated immunity (athymic) or with combined defects in innate and cellular immunity (beige athymic) were i.v. challenged with C. neoformans M7. The nonencapsulated strain of C. neoformans produced a persistent low-grade infection in the brains of all immunodeficient and immunocompetent mice used in this study. Immunocompetent mice (nu/+;bg/+) and immunodeficient bg/bg mice readily cleared nonencapsulated cryptococci from their kidneys, liver, lungs, and spleen. In contrast to nu/+ mice, nu/nu mice had a reduced capacity to clear nonencapsulated cryptococci from their kidneys and liver after i.v. challenge. Both bg/bg-nu/nu and bg/bg-nu/+ mice developed a low-grade infection in their kidneys, liver, lungs, and spleen, which was maintained throughout the 21-day study. Persistent infections were not due to reversion to an encapsulated state. These data indicate that a capsule may not always be necessary for C. neoformans to survive, in vivo, in tissues of immunodeficient and immunocompetent mice.

Role of natural killer cells in resistance to systemic cryptococcosis.

These studies demonstrate that Cryptococcus neoformans infection induced a dose-dependent augmentation of splenic natural killer (NK) cell activity by bg/+, but not bg/bg mice. To directly assess the role of NK cells in resistance to C. neoformans, bg/+ and bg/bg mice were treated with anti-NK-1.1 monoclonal antibody (mAb). Anti-NK-1.1-treatment abrogated the augmented NK cell activity observed during C. neoformans infection in bg/+ mice. Anti-NK-1.1-treated bg/+ mice had higher C. neoformans colony forming units (CFU) in their lungs on days 3 and 7 after intravenous (i.v.) challenge than control bg/+ mice. Moreover, the number of C. neoformans CFU in the lungs of anti-NK-1.1-treated bg/+ mice on days 3 and 7 were similar to those observed for infected bg/bg mice. By day 14, however, no differences in C. neoformans CFU were evident in the lungs of anti-NK-1.1-treated and control bg/+ mice. Anti-NK-1.1-treatment did not alter either the growth of C. neoformans in the spleens, livers, kidneys, or brain of bg/+ mice or the susceptibility of bg/bg mice to systemic cryptococcosis. These studies suggest that NK cells do not play a role in resistance to systemic cryptococcosis in the spleen, but do appear to play an early, but transient role in resistance to C. neoformans in the lungs. Overall, congenital defects in polymorphonuclear neutrophils (PMNs) and macrophages (M phi s), in addition to defects in NK cells, contribute to the enhanced susceptibility of bg/bg mice to systemic cryptococcosis.

Inflammatory responses to cryptococcosis in congenitally athymic mice.

Histopathology revealed that nu/nu mice developed both acute and chronic inflammatory responses following infection with Cryptococcus neoformans. In comparison to inflammatory responses in nu/+ mice, the responses in nu/nu mice were delayed, less intense, contained predominantly more polymorphonuclear leukocytes (PMNs) than macrophages (M phi s), and did not develop into granulomas. In addition, nu/nu mice developed cryptococcal skin lesions demonstrating that C. neoformans is dermatotropic in a T-cell deficient host. Quantitative culturing of infected organs confirmed that delayed and incomplete inflammatory responses observed in nu/nu mice correlated with their enhanced susceptibility to C. neoformans.