In Vitro Effects of Benzophenone-2 and Octyl-Methoxycinnamate on the Production of Interferon-γ and Interleukin-10 by Murine Splenocytes

Chemical ultraviolet light absorbers (UV-filters) are nowadays widely used in cosmetic and plastic industry. Recent in vitro and in vivo studies have reported that certain chemical UV-filters possess estrogenic activity raising the question of whether these compounds are safe to human health. Work on estrogenic effects of these compounds, however, has focused mostly on reproductive organs, and as the presence of estrogen receptors has been identified in several cells of the immune system, UV screens also may have a great impact on immunity. Thus, we have studied the in vitro effects of two widely used UV-filters—benzophenone-2 (BP-2) and octyl-methoxycinnamate (OMC)—on the production of interferon (IFN)-γ and interleukin (IL)-10, two cytokines representing Th1- and Th2-type response, respectively, by activated murine splenocytes. Cells were cultured on 48-well plastic plates and stimulated with 12-miristate 13-acetate (PMA) (5 ng/ml) and ionomycin (50 ng/ml) in the presence of different concentrations (10^?5–10^?8M) of the studied substances or 17β-estradiol (E2). After 48 hr incubation the supernatants were collected and the levels of IFN-γ and IL-10 were measured using immunoenzymatic assay. Our results show that BP-2 and OMC at high concentrations (10^?5M) shifted the Th1/Th2 balance toward a Th2 response (lower IFN-γ production and higher IL-10). These effects were comparable to those of E2. Our results clearly show that UV-screens at high doses also may possess immunomodulatory effects some of which resemble those of E2.     

Relationship Between Interferon-γ, Interleukin-10, and Interleukin-12 Production in Chronic Hepatitis C and In Vitro Effects of Interferon-α

In the current study, increased interferon (IFN)-, interleukin (IL)-10, and IL-12 p40 serum levels were observed in patients with chronic hepatitis C (CHC) compared to controls. Patients also displayed an increased spontaneous IFN- release but a deficient peripheral blood mononuclear cells (PBMC) IFN- production following stimulation with Staphylococcus aureus Cowan I strain (SAC). No difference was found with reference to spontaneous or phytohaemagglutinin (PHA)-induced IL-10 release between patients and controls, whereas a higher IL-12 p70 and IL-12 p40 secretion triggered by SAC was observed in patients. Moreover, IL-12 p40/p70 ratio following SAC stimulation was higher in patients compared to controls and a negative correlation was found between this ratio and IFN- amounts. Recombinant IL-12 (rIL-12) as well as neutralizing anti-IL-10 monoclonal antibodies (mAbs) were able to restore the compromised IFN- production. Of note, anti-IL-10 supplementation induced a lower IL-12 p40/p70 ratio in HCV subjects as compared to controls. Finally, IFN- upregulated in vitro IFN-, IL-10, and IL-12 p70 release but not IL-12 p40 secretion, this giving rise to a normalization of IL-12 p40/p70 ratio. The data suggest the occurrence of an enhanced responsiveness to IL-10 modulating effects, likely mediated by an imbalance of IL-12 p40/p70 ratio, in chronic HCV infection. Cytokine balance restoration might thus contribute to achieve therapeutical results in chronic hepatitis C.     

Interleukin-4 and Interferon-γ Inhibit Prostaglandin Production by Interleukin-1β-Stimulated Human Periodontal Ligament Fibroblasts

The purpose of the present study was to investigate the involvement of cyclooxygease-1 (COX-1) and cyclooxygenase-2 (COX-2) in prostaglandin (PG) production by human periodontal ligament (PDL) fibroblasts stimulated with a proinflammatory cytokine, inerleukin-1 (IL-1), and to examine the effect of interleukin-4 (IL-4), a Th2 cytokine, and interferon- (IFN-), a Th1 cytokine, on PG production by the cells. IL-1-stimulated PDL fibroblasts produced prostaglandin E₂ (PGE₂) in a time-dependent manner. Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE₂ production by IL-1-stimulated cells. Northern blot analysis showed that COX-2 mRNA was detected in IL-1-stimulated PDL cells, although not detected in unstimulated cells, while expression of COX-1 mRNA was in the same extent in both the cells. Dexamethasone inhibited COX-2 mRNA expression, COX activity and PGE₂ production in IL-1-stimulated cells. IL-4 and IFN- suppressed PGE₂ production by IL-1-stimulated PDL fibroblasts, but COX activity enhanced by IL-1 treatment was significantly inhibited by IL-4, not by IFN-. Northern blot analysis showed that IL-4 depressed COX-2 mRNA expression with no effect on COX-1 mRNA expression. On the other hand, IFN- had no effect on expression of COX-1 and -2 mRNA. These data suggest that COX-2 is primarily responsible for PGE₂ production by IL-1-stimulated human PDL fibroblasts and that IL-4 inhibited PGE₂ production by IL-1-stimulated PDL fibroblasts through down-regulation of COX-2 expression, while IFN- suppressed the PGE₂ production with no effect on COX-2 expression.     

In Vitro Induction of Allergen-Specific Interleukin-10-Producing Regulatory B Cell Responses by Interferon-γ in Non-Immunoglobulin E-Mediated Milk Allergy

Purpose

Specific oral immunotherapy (SOIT) using interferon-γ (IFN-γ) has been successful as a food allergy treatment. Interleukin-10 (IL-10)-producing regulatory B cells (Br1s) play a role in immune tolerance to food allergens. In addition, IFN-γ shows tolerogenic effects on allergen-induced Br1 responses.

Methods

Eleven patients that were allergic to cow''s milk and 12 milk-tolerant subjects were selected by double-blind placebo-controlled food challenge (DBPCFC) and clinical characteristics. The immunomodulatory effects of IFN-γ on allergen-specific Br1 responses were evaluated in 6 milk allergy patients and 8 milk-tolerant subjects. Peripheral blood mononuclear cells (PBMCs) from subjects were stimulated with casein and/or IFN-γ and analyzed by flow cytometry.

Results

IFN-γ had no effect on total cell counts or the proportion of Br1 cells in PBMCs. IFN-γ stimulation did not change total Br1 cell counts or the percentage of Br1s among CD5(+) B cells in the milk allergy or the milk-tolerant groups. In the milk allergy group, Br1 counts were not different between the control and the casein stimulation but significantly increased in the IFN-γ + casein stimulated cells, and the Br1 fractions were decreased after casein stimulation and recovered in the addition of IFN-γ for stimulation. In the milk-tolerant group, Br1 counts increased in the casein stimulated cells and in the IFN-γ + casein stimulated cells, but the increase was significantly less when IFN-γ was added, and the Br1 fractions were increased after casein stimulation and IFN-γ + casein stimulation, that was not significant when IFN-γ was added.

Conclusions

IFN-γ-induced allergen-specific Br1 responses in the PBMCs of milk allergy patients play a role in milk allergen-specific tolerance induction in vitro. Further investigations into the molecular immunological mechanisms underlying the induction of allergen-specific Br1 responses are needed.     

Low In Vitro Production of Interferon-γ and Tumor Necrosis Factor-α in HIV-Seronegative Patients with Pulmonary Disease Caused by Nontuberculous Mycobacteria

We studied 32 HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria (NTM). Immunologic studies included lymphocyte subset analysis by flow cytometry, measurement of interferon- (IFN-) and tumor necrosis factor- (TNF-) production followingin vitro stimulation of diluted whole blood (DWB) and peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA), anti-CD3 as well as purified protein derivative of tuberculin (PPD), and in four cases with different amounts of the very mycobacterium, which caused disease in these patients. Data were compared to those of 30 HIV-seronegative patients with disease byMycobacterium tuberculosis (MTb). Following -CD3-stimulation of PBMC, NTM patients showed lower IFN-(P < 0.00005) and lower TNF-(P < 0.02). For a subgroup of tuberculin skin test-positive NTM patients we found significantly lower PPD-induced IFN- releases in cultured DWB(P < 0.0002) and PBMC(P < 0.0004) compared to MTb patients. Data for PPD-induced TNF- release for this subgroup were also significant(P < 0.001 andP < 0.05, respectively). The four NTM patients with poor PPD-induced IFN- response hardly showed increased cytokine production on stimulation with their specific mycobacterium. The lower production capacity of IFN- and TNF- of NTM patients compared to the MTb patients points to an immunologic imbalance forming the basis for their increased susceptibility to pulmonary infections by nontuberculous mycobacteria.     

The Adenosine-Dependent Angiogenic Switch of Macrophages to an M2-Like Phenotype is Independent of Interleukin-4 Receptor Alpha (IL-4Rα) Signaling

Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A_2A receptor (A_2AR) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d.” Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα^?/? mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.     

Intracellular Distributing and Interferon-γ Secretion of Human Interleukin-18 in BxPC-3 Cells

Objective: To investigate the characteristics of interleukin-18 (IL-18) in vitro, explore IL-18, interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretive activity in BxPC-3 line cells with interleukin-18 mutants.Methods: Human IL-18 full-length gene (hIL-18-F) and the hIL-18 presumed mature protein gene (hIL-18-M) were inserted into the expression vector pEGFP-N1, to construct recombinant plasmids as Mu0, Mu1, Mu2, Mu3, and Mu4, and the recombinant plasmids were then transferred into BxPC-3 line cells. There are significant differences between Mu1, Mu2 and the pEGFP-C1 control group (P<0.05) by 3-(4,5-dimethiazol- 2-yl)- 2,5-diphenyltetrazolium bromide (MTT) for a proliferation assay, and the fluorescence of the Mu1 and Mu 2 appeared targeted to the membranous region in the BxPC-3 cells after transfected 24h by confocal laser scanning microscope (OLSM).To characterize the intracellular distribution of hIL-18, recombinant IL-18 were each fused to the enhanced green fluorescent protein gene, and expressed in BxPC-3 cells.Results: Results showed that the Mu1 tended to the membranous region in BxPC-3 cells, this indicates that the N-terminal former amino acid peptide helped ChIL-18 target to BxPC-3 cellS membranes. ELISA results demonstrated that IFN-γ and IL-18 secreted levels of BxPC-3 cells transfecting with recombinant plasmid showed an significant difference (P<0.01); refers to IL-2 expression, the two BxPC-3 cells groups transfecting with recombinant plasmid have no significant function (P>0.05).Conclusions: The results showed that hIL-18 and hIL-18 presumed mature protein can induce the secretion of IFN-γ in BxPC-3 cells, and increase the expression of IL-18, but they have no effects on IL-2.     

Dynamics of Structural Transformations of BCG Granulomas and Expression of TNF-α and Granulocyte-Macrophage CSF by Macrophages In Vitro

The spleens were isolated from mice at different times after BCG infection and BCG granulomas were explanted and cultured in vitro. Cell migration, chemoattractant potential, and expression of TNF-α and granulocyte-macrophage CSF (GM-CSF) by macrophages migrated from granulomas were evaluated in granulomas. The number of macrophages able to migrate; migrating out of granulomas, expressing TNF-α and GM-CSF decreased with increasing the time after infection. The number of cells in “dissociating” granulomas correlates with the number of macrophages containing live BCG mycobacteria in the cytoplasm.     

Transgenic Expression of Interferon-γ in Mouse Stomach Leads to Inflammation,Metaplasia, and Dysplasia

Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H⁺/K⁺ ATPase β promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear β-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion.Gastric adenocarcinoma is one of the most common causes of cancer-related deaths worldwide,¹ and although the incidence of these cancers in the United States is decreasing, the 5-year survival rate is a dismal 27%.² Gastric cancer in humans develops through a series of stages, first defined by Correa and Piazuelo, which includes gastritis, atrophy, intestinal metaplasia, dysplasia, and carcinoma.³ Infection with Helicobacter pylori is the greatest single risk factor for the development of gastric cancer.⁴ The gastritis that accompanies Helicobacter infection plays a major role in gastric cancer development⁵; however, the key inflammatory mediators driving this process have not been fully defined. Data from several types of malignancy point to critical functions for inflammation in cancer,⁶ with an interplay between extrinsic factors (infection/inflammation) and intrinsic factors (oncogenes/tumor suppressor genes) driving neoplastic progression.⁷ Targeting pivotal pro-tumorigenic cytokines may thus be useful for the treatment or prevention of certain types of cancer.⁸Interaction of Helicobacter with epithelial cells in the gastric corpus triggers an immune response that leads to the production of multiple cytokines and the establishment of chronic inflammation. Polymorphisms in the IL-1β gene predispose to gastric cancer development in humans,^9,10 and overexpression of IL1β in the corpus of transgenic mice leads to gastric dysplasia and cancer,¹¹ pointing to an important role for this cytokine in gastric tumorigenesis. A Th1-polarized immune response, characterized by elevated expression of interferon-γ (IFN-γ), is activated in Helicobacter-infected stomach and appears to be required for preneoplastic changes, including parietal cell atrophy and the development of gastric metaplasia known as spasmolytic peptide-expressing metaplasia (SPEM),^12–14 which may be a precursor to gastric cancer.¹⁵In mice, transgene-driven expression of IFN-γ in the brain leads to robust induction of the Hh pathway ligand/activator Sonic hedgehog (Shh). This deregulated activation of the Hh signaling pathway in turn leads to the development of medulloblastoma,^16,17 an Hh pathway–driven brain tumor. Because elevated activity of the Hedgehog (Hh) signaling pathway has also been linked to gastric cancer,^18,19 these results raised the interesting possibility that IFN-γ may contribute to inflammation-associated gastric neoplasia in part through induction of Shh expression and oncogenic signaling activity.We tested the potential role of IFN-γ as a pivotal cytokine driving neoplastic progression in stomach by generating transgenic mice, designated H/K-IFN-γ, which overexpress murine IFN-γ driven by an H/K ATPase β promoter, which targets transgene expression to the gastric corpus. We show that IFN-γ overexpression leads to inflammation, increased proliferation, parietal cell and chief cell atrophy, SPEM, an increased number of myofibroblasts, dysplasia, and, in a subset of mice, tumor development. Interestingly, while this work was underway, another group produced mice with stomach-targeted overexpression of IFN-γ. Those mice, in which IFN-γ signaling was activated at lower levels than the mice described here, did not exhibit gastritis and SPEM. Indeed, these processes, as well as neoplasia driven by Helicobacter infection or IL-1β overexpression, were blocked.²⁰ In striking contrast, we show that robust expression of IFN-γ in our model is sufficient to orchestrate multiple changes in inflammatory, epithelial, and mesenchymal cell populations to drive premalignant progression in stomach, though additional alterations appear to be required for the development of full-blown neoplasia.     

Effect of Plasma Lipoproteins and Their Complexes with Polysaccharides on Interleukin-1β Concentration in Macrophages of Mice with HA-1 Ascitic Hepatoma

Experiments on cultured peritoneal macrophage from mice with HA-1 ascitic hepatoma showed that plasma lipoproteins present in the incubation medium decreased intracellular concentration of interleukin-1β. These changes were most pronounced for high-density lipoproteins (alone or in combination with cortisol). Bacterial and yeast polysaccharides had little effect on interleukin-1β concentration in macrophages. Addition of polysaccharides in combination with lipoproteins was followed by a 2-3-fold decrease in interleukin-1β concentration. A combination of polysaccharides and high-density lipoproteins had the strongest effect. These properties of plasma lipoproteins should be taken into account in the correction of macrophage function during tumor growth.     

IFN-γ Inhibits the Suppressive Effects of PGE2 on the Production of Tumor Necrosis Factor-α by Mouse Macrophages

Tumor necrosis factor-α (TNF-α) has become known as a central mediator of responses to endotoxin, rheumatoid diseases, and other forms of inflammation. Current investigations indicate that the production of TNF-α is controlled by other mediators, including interferon-γ (IFN-γ) and prostaglandin E₂ (PGE₂). In the present study, we investigated the regulatory effects of IFN-γ and/or PGE₂ on LPS-induced TNF-α production and mRNA expression in mouse peritoneal macrophages using the enzyme immunoassay and Northern blot analysis, respectively. In response to 10 ng/ml of LPS, TNF-α production reached a maximum at approximately 4 hrs, followed by rapid decline. At the molecular level, TNF-α mRNA accumulated rapidly after LPS exposure, reaching a peak by 3 hr, and declined more rapidly than did the production of TNF-α. Exposure of macrophages to 100 U/ml of IFN-γ caused an increase in both the TNF-α production and mRNA expression induced by LPS. Exogenous PGE₂ caused a dose dependent reduction in LPS-induced TNF-α mRNA accumulation as well as TNF-α production. Macrophages primed with IFN-γ showed the reduced responsiveness to the suppressive effect of PGE₂ on the production of TNF-α and the accumulation of TNF-α mRNA. These findings indicate that the suppressive effects induced by PGE₂ on the accumulation of TNF-α mRNA as well as the production of TNF-α can be reduced by the pretreatment of macrophages with IFN-γ. These studies demonstrate the role of IFN-γ as an immunomodulating compound that may effectively regulate TNF-α production by modulation of macrophage responsiveness to PGE₂.     

Expressions of Interleukin-1β and Interleukin-6 Within Aortas and Uteri of Rats with Various Severities of Ligature-Induced Periodontitis

To investigate the associations of periodontitis with histological lesions in some other organs, various severities of periodontitis were induced in rats by 3/0 silk ligatures tied around different numbers of their molar necks. Six weeks after the initial placement of ligatures, all rats were sacrificed by an anaesthetic overdose. The distances from the cemento-enamel junction to the alveolar bone crest within the placement zone of the ligature and their contralateral zone in groups L₂ and L₃ were measured. The levels of interleukin (IL)-1β and IL-6 in serum were assayed by enzyme-linked immunosorbent assay techniques, and those within aortas and uteri were measured by real-time polymerase chain reaction and by immunohistochemistry. We divided the ligature-induced periodontitis models into mild, moderate and severe rat periodontitis and observed that although no association between periodontitis and the serum concentrations of IL-1β was detected, the differences in the severity of rat periodontitis led to varying degrees of elevated expressions of IL-1β and IL-6 within aortas and uteri.     

Critical Role for Interleukin-1β (IL-1β) during Chlamydia muridarum Genital Infection and Bacterial Replication-Independent Secretion of IL-1β in Mouse Macrophages

Recent findings have implicated interleukin-1β (IL-1β) as an important mediator of the inflammatory response in the female genital tract during chlamydial infection. But how IL-1β is produced and its specific role in infection and pathology are unclear. Therefore, our goal was to determine the functional consequences and cellular sources of IL-1β expression during a chlamydial genital infection. In the present study, IL-1β^−/− mice exhibited delayed chlamydial clearance and decreased frequency of hydrosalpinx compared to wild-type (WT) mice, implying an important role for IL-1β both in the clearance of infection and in the mediation of oviduct pathology. At the peak of IL-1β secretion in WT mice, the major producers of IL-1β in vivo are F4/80⁺ macrophages and GR-1⁺ neutrophils, but not CD45⁻ epithelial cells. Although elicited mouse macrophages infected with Chlamydia muridarum in vitro secrete minimal IL-1β, in vitro prestimulation of macrophages by Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) purified from Escherichia coli or C. trachomatis L2 prior to infection greatly enhanced secretion of IL-1β from these cells. By using LPS-primed macrophages as a model system, it was determined that IL-1β secretion was dependent on caspase-1, potassium efflux, and the activity of serine proteases. Significantly, chlamydia-induced IL-1β secretion in macrophages required bacterial viability but not growth. Our findings demonstrate that IL-1β secreted by macrophages and neutrophils has important effects in vivo during chlamydial infection. Additionally, prestimulation of macrophages by chlamydial TLR ligands may account for the elevated levels of pro-IL-1β mRNA observed in vivo in this cell type.The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of sexually transmitted infection worldwide. Infection can lead to oviduct pathology and infertility in females. Cells respond to infection with C. trachomatis by upregulating a wide assortment of genes, including those for proinflammatory cytokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6 (34, 54, 55, 68). The cellular paradigm of chlamydial pathogenesis states that tissue damage resulting from chlamydial infection is caused by excessive production of these proinflammatory cytokines (61). In support of this theory, caspase-1 knockout (KO) and Toll-like receptor 2 (TLR2) KO mice exhibit less upper genital tract pathology than wild-type (WT) mice, despite equivalent courses of infection (12, 15). This finding is noteworthy because caspase-1 KO mice lack the protease required to activate IL-1β (32) and TLR2 KO fibroblasts express less mRNA for IL-1β and lower levels of other inflammatory cytokines than WT fibroblasts during in vitro chlamydial infections (15). Additionally, administration of an IL-1β antagonist prevents C. trachomatis-induced cytotoxicity in a human fallopian tube organ culture model (30). Overall, these observations suggest that IL-1β may be a potentially important factor in the development of oviduct pathology, underscoring the need to define the role of IL-1β during in vivo chlamydial infections and to mechanistically determine how the IL-1β-converting enzyme caspase-1 is activated upon contact with Chlamydia spp. to produce mature IL-1β.Caspase-1 is the central component of the host “inflammasome” (reviewed in reference 38) and exists at rest as a 45-kDa zymogen. When activated by proteolysis, it forms a tetramer consisting of two 10- to 20-kDa heterodimers (65), with the 20-kDa portion containing the active-site cysteine necessary to cleave the cytokine precursors pro-IL-1β and pro-IL-18 and also the TH2 cytokine precursor pro-IL-33 (10, 24, 59, 63). Caspase-1 has many other substrates in the cell (60), but its two main targets relevant to inflammation are IL-1β (10) and IL-18 (24). Both IL-1β and IL-18 require this processing for biologic activity. IL-18 plays a role in stimulating gamma interferon production from T cells (18, 56) and natural killer cells (11), suggesting a protective role for IL-18 during genital chlamydial infection. This leads to the theory that the detrimental role of caspase-1 for oviduct pathology during chlamydial infection is mediated mainly via overproduction of the proinflammatory cytokine IL-1β.Several studies have shown that caspase-1 activation during intracellular bacterial infection involves active contribution from the bacteria. For instance, activation occurs following bacterial type III secretion (T3S)-dependent introduction into the cytosol of Yersinia pestis YopJ (35), Pseudomonas aeruginosa flagellin (42), or Salmonella enterica flagellin (20). Early work examining the role of caspase-1 during chlamydial infection by using HeLa cells demonstrated that the activation of caspase-1 late in the infection cycle (36 to 48 h postinfection) is dependent on both chlamydial viability and protein synthesis (36). Additionally, the T3S antagonist INP0007 prevents activation of caspase-1 in C. trachomatis-infected HeLa cells (66). Low-level production of IL-1β was abolished in caspase-1 KO mouse peritoneal macrophages infected in vitro with mouse chlamydia C. muridarum (12), which can replicate effectively in mouse macrophages (46). However, it is not known if the infected epithelial cells or the inflammatory cells recruited to the site of infection are the main producers of IL-1β during an in vivo genital infection. Furthermore, the specific role for IL-1β during in vivo genital infection has not been addressed. In this study, we show that IL-1β is required for optimal chlamydial clearance but also contributes to the development of genital tract pathology. Additionally, macrophages and neutrophils and not epithelial cells account for the high expression levels of IL-1β in vivo. We further describe mechanistic details of IL-1β production by using in vitro infections of lipopolysaccharide (LPS)-primed macrophages. Overall, these macrophages produced IL-1β by both caspase-1- and serine protease-dependent mechanisms, but surprisingly, IL-β secretion by these cells occurred in a bacterial protein synthesis-independent manner, although chlamydial viability was required.     

Distinct Tumor Necrosis Factor-α Responses in Alveolar and Peritoneal Macrophages Are Associated with Local Levels of Endotoxin

Tumor necrosis factor alpha (TNF-) responses of alveolar macrophages (AMs) and peritoneal macrophages (PMs) were studied in rats after intravenous injection of lipopolysaccharide (LPS). High levels of plasma TNF-, increased pulmonary myeloperoxidase activity, and leukopenia occurred within 2 h after LPS injection. Alveolar spaces exhibited a strict compartment property, as manifested by only slightly increased LPS and TNF- levels in alveolar lavage fluid and an unchanged capacity of AMs to produce TNF-. By contrast, the peritoneal cavity had greatly increased local LPS and TNF- levels and a diminished PMs TNF- response to LPS. The amount of LPS in the alveolar spaces was less than 0.2% of the level in peritoneal fluid. These results indicate that activation of resident macrophages is dependent on the amounts of local LPS and, in addition, suggest that resident AMs neither participate in the plasma TNF- response nor contribute to neutrophil sequestration in the lung during the early stages of endotoxemia.     

Interleukin-32γ Enhances the Production of IL-6 and IL-8 in Fibroblast-Like Synoviocytes Via Erk1/2 Activation

Introduction  

In the present study, we examined the effect of the pro-inflammatory cytokine IL-32γ, the most biologically active isoform, and its related molecules in fibroblast-like synoviocytes (FLS).