Vasoactive intestinal peptide balances pro- and anti-inflammatory cytokines in the Pseudomonas aeruginosa-infected cornea and protects against corneal perforation

Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-gamma, IL-1beta, MIP-2, and TNF-alpha, whereas anti-inflammatory mediators, IL-10 and TGF-beta1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mphi) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mphi from both mouse strains resulted in decreased IL-1beta and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mphi/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.     

Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation

The kinetics of IL-1 (alpha and beta) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1alpha and -1beta (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i. ). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1alpha and -1beta (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1beta was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1beta polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.     

Macrophages restrict Pseudomonas aeruginosa growth,regulate polymorphonuclear neutrophil influx,and balance pro- and anti-inflammatory cytokines in BALB/c mice

The role of macrophages in Pseudomonas aeruginosa corneal infection in susceptible (cornea perforates), C57BL/6 (B6) vs resistant (cornea heals), BALB/c mice was tested by depleting macrophages using subconjunctival injections of clodronate-containing liposomes before corneal infection. Both groups of inbred mice treated with clodronate-liposomes compared with PBS-liposomes (controls) exhibited more severe disease. In B6 mice, the cornea perforated and the eye became extremely shrunken, whereas in BALB/c mice, the cornea perforated rather than healed. The myeloperoxidase assay detected significantly more PMN in the cornea of both groups of mice treated with clodronate-liposomes vs PBS-liposomes. In independent experiments, ELISA analysis showed that protein levels for IL-1 beta, macrophage-inflammatory protein 2, and macrophage-inflammatory protein 1 alpha, all regulators of PMN chemotaxis, also were elevated in both groups of mice treated with clodronate-liposomes. Bacterial plate counts in B6 mice treated with clodronate-liposomes were unchanged at 3 days and were higher in control-treated mice at 5 days postinfection (p.i.), whereas in BALB/c mice, bacterial load was significantly elevated in the cornea of mice treated with clodronate-liposomes at both 3 and 5 days p.i. mRNA expression levels for pro (IFN-gamma and TNF-alpha)- and anti (IL-4 and IL-10)-inflammatory cytokines also were determined in BALB/c mice treated with clodronate-liposomes vs control-treated mice. Expression levels for IFN-gamma were significantly elevated in mice treated with clodronate-liposomes at 3 and 5 days p.i., while IL-10 levels (mRNA and protein) were reduced. These data provide evidence that macrophages control resistance to P. aeruginosa corneal infection through regulation of PMN number, bacterial killing and balancing pro- and anti-inflammatory cytokine levels.     

IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice. Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.     

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.     

Pathogenic mechanisms of P. aeruginosa keratitis: a review of the role of T cells,Langerhans cells,PMN, and cytokines

The aim of this article is to review our current understanding of the role of cytokines, chemokines, T cells, Langerhans cells, and neutrophils (PMN) and their interactions in vivo in the host response to Pseudomonas aeruginosa ocular challenge. The cellular/cytokine network in vivo has begun to be unraveled, and the data discussed provide substantive evidence for a regulatory role of CD4(+) T cells (Th1 type) contributing directly to persistence of PMN in the cornea of susceptible C57BL/6 (cornea perforates) versus resistant BALB/c (cornea heals) mice. Additionally, in the susceptible mouse model, CD4(+) T cells interact with Langerhans cells and B7/CD28 ligation appears critical for antigen presentation and the susceptibility response. Various cytokines and chemokines (e.g., MIP-1alpha, IL-1beta, MIP-2, IL-12, and IFN-gamma) and their pattern of sustained upregulation after infection in susceptible versus resistant mice also will be discussed in light of an in vivo cytokine network. T-cell-mediated pathogenic mechanisms are of importance in development of the susceptible response to P. aeruginosa ocular infection. In the absence of T-cell infiltration into the cornea, PMN do not persist in the stroma, and cytokines and chemokines are better balanced, resulting in decreased stromal destruction and the resistance response.     

The importance of IL-1 beta and TNF-alpha,and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.     

Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1alphabeta, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-alpha was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1alpha and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1alpha or IL-1beta and was partially blocked by injection of neutralizing Ab against IL-1alpha but not IL-1beta. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1beta were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1alpha or IL-1beta (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1beta. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.     

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

Contribution of the cornea to cytokine levels in the whole eye induced during the early phase of Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome, and is regulated by cytokines produced in the ocular tissue. In this study, we assessed the relative contribution of the cytokines produced in the cornea to the inflammatory response of the whole eye to gain a better understanding of the inflammatory and regulatory processes in the ocular environment during localized corneal infection. C57BL/6 mice were challenged by topical application of P. aeruginosa to wounded corneas. Corneas and whole eyes were harvested 24 h post-challenge and bacterial numbers, myeloperoxidase levels and the levels of cytokines known to be important in keratitis were determined. The site of production of IL-6 and KC in the retina was determined by in situ hybridization. Before infection, 90% of macrophage inflammatory protein (MIP)-2 and approximately 80% of all IFN-gamma and IL-10 produced constitutively in the eye was found outside the cornea. Twenty-four hours after infection, bacterial numbers, levels of myeloperoxidase, and levels of MIP-2 and IL-1 were not different, whether measured in cornea or whole eye. However, expression of IL-6, KC, IFN-gamma and IL-10 was significantly greater in whole eyes than in the corneas of infected eyes. The cells expressing IL-6 and KC in the retina were identified by in situ hybridization. This study indicates that during corneal inflammation, the response of the whole eye as well as the cornea needs to be considered.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma-inducible protein 10 (IP-10) and monocyte interferon-gamma-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.     

CXC chemokine receptor 2 but not C-C chemokine receptor 1 expression is essential for neutrophil recruitment to the cornea in helminth-mediated keratitis (river blindness)

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2(-/-) and CCR1(-/-) mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1 alpha was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2(-/-) mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2(-/-) mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2(-/-) mice, which was similar to BALB/c mice. Furthermore, although MIP-1 alpha production was lower in CXCR2(-/-) mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.     

Toll-like receptor signaling and SIGIRR in renal fibrosis upon unilateral ureteral obstruction

Innate immune activation via IL-1R or Toll-like receptors (TLR) contibutes to acute kidney injury but its role in tissue remodeling during chronic kidney disease is unclear. SIGIRR is an inhibitor of TLR-induced cytokine and chemokine expression in intrarenal immune cells, therefore, we hypothesized that Sigirr-deficiency would aggravate postobstructive renal fibrosis. The expression of TLRs as well as endogenous TLR agonists increased within six days after UUO in obstructed compared to unobstructed kidneys while SIGIRR itself was downregulated by day 10. However, lack of SIGIRR did not affect the intrarenal mRNA expression of proinflammatory and profibrotic mediators as well as the numbers of intrarenal macrophages and T cells or morphometric markers of tubular atrophy and interstitial fibrosis. Because SIGIRR is known to block TLR/IL-1R signaling at the level of the intracellular adaptor molecule MyD88 UUO experiments were also performed in mice deficient for either MyD88, TLR2 or TLR9. After UUO there was no significant change of tubular interstitial damage and interstitial fibrosis in neither of these mice compared to wildtype counterparts. Additional in-vitro studies with CD90+ renal fibroblasts revealed that TLR agonists induce the expression of IL-6 and MCP-1/CCL2 but not of TGF-β, collagen-1α or smooth muscle actin. Together, postobstructive renal interstitial fibrosis and tubular atrophy develop independent of SIGIRR, TLR2, TLR9, and MyD88. These data argue against a significant role of these molecules in renal fibrosis.     

Differences in interleukin-12 and -15 production by dendritic cells at the early stage of Listeria monocytogenes infection between BALB/c and C57 BL/6 mice

The mechanisms responsible for the resistance of C57BL/6 mice and for the susceptibility of BALB/c mice to infection with Listeria monocytogenes were studied by comparing early IL-12 and IL-15 production by dendritic cells (DC) after infection with L. monocytogenes. Splenic DC expressing CD11b(low) and CD11c(+) obtained from C57BL/6 mice at 3 and 6 h after L. monocytogenes infection expressed higher levels of IL-12 p40 mRNA and IL-12 p40 protein than did those from BALB/c mice. Concurrently, a larger amount of IFN-gamma was produced by the splenic T cells from C57BL/6 mice in response to immobilized anti-TCRalphabeta mAb than by those from BALB/c mice, while the splenic T cells from BALB/c mice produced a higher level of IL-4 upon TCR alphabeta stimulation than did those of C57BL/6 mice. IL-15 mRNA and intracellular IL-15 protein were detected more abundantly in the DC from C57BL/6 mice than in those from BALB/c mice on day 3 after infection. CD3(+) IL2Rbeta (+) cells in the spleen were increased in C57BL/6 mice but not in BALB/c mice at the early stage after infection. Furthermore, IL-12Rbeta2 gene expression was up-regulated in T cells from C57BL/6 mice but not in those from BALB/c mice at the early stage after listerial infection. These results suggest that the difference in early production of IL-12 and IL-15 by DC may at least partly underlie the difference in susceptibility to L. monocytogenes between C57BL/6 and BALB/c mice.