Cytokines In Peritoneal Macrophages Research Articles

The Macrophage Activator GcMAF-RF Enhances the Antitumor Effect of Karanahan Technology through Induction of M2-M1 Macrophage Reprogramming.

Macrophages are the immune cells of high-immunological plasticity, which can exert both pro- and anti-inflammatory activity, as well as repolarize their phenotype to the opposite or neutral one. In this regard, M2 macrophages of the tumor-associated stroma (TAS) are a promising therapeutic target in treating malignant neoplasms. Using FACS assay, we have estimated the CD11b+/Ly-6G+/Ly-6C+ fraction of macrophages from the peritoneum and TAS in intact healthy mice and those with developed Lewis carcinoma, both untreated and treated according to Karanahan technology in combination with group-specific macrophage activator (GcMAF-RF). As well, the pattern of pro- and anti-inflammatory cytokines mRNA expression in different groups of experimental and tumor-bearing animals was assessed. It was found that: (i) exposure of intact mice to GcMAF-RF results in the increased number of CD11b+/Ly-6C+ peritoneal macrophages and, at the same time, the expression pattern of cytokines in peritoneal macrophages switches from that characteristic of the mixed M1/M2 phenotype to that characteristic of the neutral M0 one; (ii) combination of Karanahan technology and GcMAF-RF treatment results in M0/M1 repolarization of TAS macrophages; (iii) in tumor-bearing mice, the response of peritoneal macrophages to such a treatment is associated with the induction of anti-inflammatory reaction, which is opposite to that in TAS macrophages.

Protective effects of S100A8 on sepsis mortality: Links to sepsis risk in obesity and diabetes.

Obesity and diabetes are independent risk factors for death during sepsis. S100A8, an alarmin, is related to inflammation, obesity, and diabetes. Here, we examine the role of S100A8 in sepsis of obesity and diabetes models. Injection of S100A8 prolongs the survival of septic mice induced by lethal endotoxemia, Escherichia coli injection, or cecal ligation and puncture. S100A8 decrease the LPS-induced expression of proinflammatory cytokines in peritoneal macrophages by inhibiting TLR4-mediated signals in an autocrine manner. db/db, ob/ob, and western diet-fed mice demonstrate reduced upregulation of S100A8 induced by LPS treatment in both serum and peritoneal cells. These mice also show shorter survival after LPS injection, and S100A8 supplementation prolonged the survival. While myelomonocytic cells-specific S100A8-deficient mice (Lyz2 cre :S100A8 floxed/floxed ) exhibit shorter survival after LPS treatment, S100A8 supplementation prolonged the survival. Thus, myelomonocytic cell-derived S100A8 is crucial for protection from sepsis, and S100A8 supplementation improves sepsis, particularly in mice with obesity and diabetes.

Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1.

Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation. HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates. Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1-/-) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice. The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.

Systemic Candidiasis and TLR2 Agonist Exposure Impact the Antifungal Response of Hematopoietic Stem and Progenitor Cells.

We have previously demonstrated that Candida albicans induces differentiation of hematopoietic stem and progenitor cells (HSPCs) toward the myeloid lineage both in vitro and in vivo in a TLR2- and Dectin-1-dependent manner, giving rise to functional macrophages. In this work, we used an ex vivo model to investigate the functional consequences for macrophages derived from HSPCs in vivo-exposed to Pam3CSK4 (a TLR2 agonist) or C. albicans infection. Short in vivo treatment of mice with Pam3CSK4 results in a tolerized phenotype of ex vivo HSPC-derived macrophages, whereas an extended Pam3CSK4 treatment confers a trained phenotype. Early during candidiasis, HSPCs give rise to macrophages trained in their response to Pam3CSK4 and with an increased fungicidal activity; however, as the infection progresses to higher fungal burden, HSPC-derived macrophages become tolerized, while their fungicidal capacity is maintained. These results demonstrate that memory-like innate immune responses, already described for monocytes and macrophages, also take place in HSPCs. Interestingly, extended Pam3CSK4 treatment leads to an expansion of spleen HSPCs and myeloid cells, and drastically reduces the fungal burden in the kidney and spleen during systemic C. albicans infection. This protection against tissue invasion is abrogated by immunodepletion of HSPCs, suggesting their protective role against infection in this model. In addition, HSPCs produce in vitro cytokines and chemokines in response to C. albicans and Pam3CSK4, and these secretomes are capable of inducing myeloid differentiation of HSPCs and modulating peritoneal macrophage cytokine responses. Taken together, these data assign an active role for HSPCs in sensing pathogens during infection and in contributing to host protection by diverse mechanisms.

Tofogliflozin Suppresses the Progression of Atherosclerosis and Reduces Inflammation in Macrophages of STZ-Diabetic ApoE KO Mice due to Improvement of Glucose Tolerance

Background: Selective sodium-glucose cotransporter 2 inhibitor (SGLT2i) has become widely used for treatment of diabetes. Recently, EMPA-REG OUTCOME and CANVAS Program showed that SGLT2i reduce cardiovascular events in patients with type 2 diabetes, but the mechanism still remains uncertain. We investigated the mechanisms of vascular protection of Tofogliflozin (Tofo), one of the SGLT2i. Method: 1) Streptozotocin (STZ)-diabetic apolipoprotein-E-deficient (ApoE KO) mice were separated into two groups, one group were fed normal chow and the other were fed normal chow containing 0.005% Tofo for ad libitum. After 6weeks, all mice were administered thioglycolate intraperitoneally, and 4 days later, peritoneal macrophages were collected to investigate the expressions of inflammatory cytokines of peritoneal macrophages by real-time PCR. Histological analyses were carried out by Sudan IV, Oil Red O, and MOMA-2 staining. 2) The same experiment targeted were carried out towards nondiabetic ApoE KO mice. Results: 1) Tofo increased food and water intake and decreased blood glucose, but there were no significant changes in body weight, serum levels of free fatty acid, triglyceride and total cholesterol between two groups. The legions of atherosclerosis and macrophages accumulations were significantly suppressed in Tofo group. Moreover, the expression levels of TNF-α, IL-β were significantly suppressed in macrophages from Tofo group. 2) Nondiabetic ApoE KO mice showed similar increase in food and water intake, but failed to exhibit differences in blood glucose, Sudan IV staining, and expressions of inflammatory cytokines in macrophages, as observed in diabetic ApoE KO mice. Conclusion: Tofogliflozin suppresses the progression of atherosclerosis and reduces inflammation in macrophages of STZ- diabetic ApoE KO mice. These beneficial effects may be exerted due to improvement of glucose tolerance. Disclosure M. Iwamoto: None. N. Kubota: None. T. Kubota: None. Y. Sakurai: None. N. Wada: None. I. Takamoto: None. S. Shioda: None. T. Kadowaki: Consultant; Self; Novo Nordisk A/S, AstraZeneca, Merck Sharp & Dohme Corp.. Research Support; Self; Kissei Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Sanofi, Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Astellas Pharma, Daiichi Sankyo Company, Limited, Takeda, Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Astellas Pharma, AstraZeneca, Merck Sharp & Dohme Corp., Ono Pharmaceutical Co., Ltd., Takeda, Eli Lilly and Company, Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk A/S.

Myricetin attenuated LPS induced cardiac injury in vivo and in vitro.

Sepsis induced myocardial dysfunction (SIMD) is a common complication and leads to an increased mortality. SIMD is closely related to inflammation and oxidative stress. Myricetin exhibits strong capacities of anti-inflammation and anti-oxidative stress, but its pharmacological effects for lipopolysaccharide (LPS) induced cardiac injury remains undefined. This study aimed to explore whether myricetin was efficient to alleviate SIMD in mice and neonatal rat cardiomyocytes injury. Mice administrated with myricetin (100mg/kg, po, bid) or vehicle groups were challenged with LPS (10mg/kg, ip), and cardiac functions examined by echocardiography after 12hr LPS exposure. LPS markedly impaired mouse cardiac functions, which were significantly attenuated by myricetin administration. Myricetin significantly reduced the production of inflammatory cytokines both in serum and cardiac tissue. Myricetin could inhibit the nuclear translocation of p65, degradation of IκBα, and cellular apoptosis in vivo and in vitro. Myricetin also prevented overexpression of iNOS and reduction of oxidoreductase (SOD and GPx) activity. Besides, Myricetin treatment could attenuate production of inflammatory cytokines of peritoneal macrophages stimulated with LPS in vitro. Thus we concluded that myricetin could attenuate the LPS induced cardiac inflammation injury in vivo and in vitro. Myricetin may be a potential therapy or adjuvant therapy for SIMD.

Therapeutic action of 5-HT3 receptor antagonists targeting peritoneal macrophages in post-operative ileus.

Post-operative ileus (POI) is induced by intestinal inflammation. Here, we aimed to clarify the effects of 5-HT3 receptor antagonists against POI. We administered three 5-HT3 receptor antagonists, ondansetron, tropisetron and palonosetron, to a mouse model of POI induced by surgical intestinal manipulation (IM). Immunohistochemistry, intestinal transit, inflammatory mediator mRNA expression and 5-HT content were measured. In some experiments, 5-HT3 A receptor null mice were used. Three 5-HT3 receptor antagonists reduced IM-induced infiltration of inflammatory CD68-positive macrophages and myeloperoxidase-stained neutrophils. Ondansetron exhibited no anti-inflammatory actions in 5-HT3 A receptor null mice. Ondansetron inhibited expression of the chemokine CCL2, IL-1β, IL-6, TNF-α and iNOS mRNAs up-regulated by IM, and also ameliorated the delayed gastrointestinal transit. Peritoneal macrophages, but not most infiltrating monocyte-derived macrophages, expressed 5-HT3 receptors. IM stimulation increased the 5-HT content of peritoneal lavage fluid, which up-regulated mRNA expression of proinflammatory cytokines in peritoneal macrophages. Immunohistochemical localization of 5-HT3 receptors suggests that ondansetron suppressed expression of these mRNAs in activated peritoneal macrophages, adhering to the serosal region of the inflamed intestinal wall. 5-HT3 receptor antagonists were anti-inflammatory, mainly targeting peritoneal macrophages expressing these receptors. They also restored the delayed gastrointestinal transit by IM. 5-HT3 receptor antagonists should be therapeutically useful agents against POI.

Saquinavir-NO inhibits IL-6 production in macrophages.

Covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a new chemical entity, named Saquinavir-NO, (Saq-NO) with reduced toxicity and potent immunoregulatory influence on T lymphocytes. In this study, we have compared head-to-head the effects of Saq-NO and Saq on mouse and rat peritoneal macrophage cytokine secretion and NO production upon in vitro, ex vivo and in vivo conditions. The results demonstrate that Saq-NO, but not Saq, potently decreased interleukin (IL)-10, IL-6 and nitrite accumulation and increased the levels of IL-1β and tumour necrosis factor (TNF) in supernatants of mouse and rat macrophage cultures in vitro. Treatment of mice with Saq-NO, but not Saq, inhibited ex vivo secretion of IL-6 from macrophages. Consistent with these findings, Saq-NO also reduced blood levels of IL-6 in lipopolysaccharide-treated mice. The observed inhibitory influence of Saq-NO on IL-6 generation in macrophages may be involved in the observed antitumour and immunomodulatory effects of the drug.

Abstract 82: Macrophage Specific ABCA1 Deletion Does Not Worsen Insulin Resistance in High Fat Diet Induced or Genetic Obese Mouse Models

Introduction Obesity associated low-grade, chronic inflammation underlies the pathogenesis of type 2 diabetes and insulin resistance. ATP-binding cassette transporter A1 (ABCA1), a membrane lipid transporter, forms nascent HDL particles by effluxing cellular free cholesterol (FC) and phospholipids to lipid-poor apolipoprotein A-I. In macrophages, ABCA1 also dampens pro-inflammatory MyD88-dependent Toll-like receptor signaling by reducing cellular membrane FC and lipid raft content, indicating a role for ABCA1 in macrophage-mediated innate immunity. However, the role of ABCA1 in macrophage-associated innate immunity in insulin resistance is unclear. Hypothesis We tested the hypothesis that macrophage-specific ABCA1 deletion may exacerbate insulin resistance by increasing obesity-induced chronic, low-grade inflammation. Methods and Results Wild type (WT) and macrophage-specific ABCA1 knockout (MSKO) mice in the C57/BL6 background were fed a 45% High Fat Diet (HFD) for 16-24 weeks or crossed into the leptin deficient (ob/ob) background to induce obesity. Our results showed that MSKO and WT mice developed obesity, insulin resistance, hypercholesterolemia, and hepatic steatosis to a similar extent with HFD feeding or after crossed into the ob/ob background. Resident peritoneal macrophages from HFD-fed or leptin deficient MSKO vs. WT mice accumulated significantly more cholesterol. Relative to chow, HFD markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissues in WT and MSKO mice. Among the examined pro-inflammatory cytokines in peritoneal macrophages, only IL-6 was highly up-regulated in ob/ob MSKO vs. WT control mice, indicating that lack of ABCA1 in macrophages does not significantly affect obesity associated chronic inflammation. Conclusion Although macrophage specific ABCA1 deficiency enhances Toll like receptor signaling, it does not significantly exacerbate obesity associated low grade, chronic inflammation, therefore has no impact on the pathogenesis of insulin resistance in both HFD induced and genetic obese mouse models.

Abstract 455: Decreased Experimental Hypertension and Inflammation in the Absence of Prolylendopeptidase Activity

Objective: Previously, we showed that inhibition of prolyloligopeptidase eliminated the differences in macrophage TNFα expression and BP response in mice lacking N-domain activity of ACE. Thus, functional studies of BP and inflammatory response in a model lacking prolylendopeptidase activity are performed. Results: After a 14 day high-dose AngII infusion (980 ng/kg-min), POP-KO(n=12) BP was 130±3 mmHg and 147±4 mmHg for POP-WT(n=12). After 14 days of low-dose AngII infusion(490 ng/kg-min), POP-KO(n=8) BP was 126±4 mmHg and 139±3 mmHg for POP-WT(n=8). To investigate differences in the inflammatory response, we examined peritoneal macrophage cytokine expression in response to AngII. The percentage of F4/80+/TNFαhigh cells vs. total F4/80+ was 17%±4.7% for POP-KO (n=8) and 49%±2.8% for POP-WT (n=8)[p<0.005]. The percentage of F4/80+/IL-6high cells vs. total F4/80+ was 15%±3.2% for POP-KO(n=8) and 39%±4.8% for POP-WT(n=8)[p<0.01]. To investigate whether there were baseline differences, TNFalpha secretion was measured in POP-KO(8%) vs. POP-WT(16%) without AngII stimulation. Baseline bone marrow analysis by flow shows 48% CD11b+Ly6ghi cells in POP-KO (n=6) vs. 39% in POP-WT (n=4). Moreover, F4/80hiLy6cintermediate cells were 1.19% in the POP-KO (n=6) vs. 1.75% in POP-WT(n=4). Conclusion: Inflammation is an emerging player in hypertension and prolylendopeptidase inhibition may play a role in reducing that inflammation. In the POP-KO mouse model, baseline differences in quantity and quality of inflammatory cells may contribute to the phenotypic differences found in vivo .

PROLYL‐OLIGOPEPTIDASE INHIBITION AMELIORATES EXPERIMENTAL HYPERTENSION AND DECREASES INFLAMMATORY CYTOKINES IN MICE LACKING ANGIOTENSIN CONVERTING ENZYME N DOMAIN ACTIVITY

Using gene targeting, ACE domain‐specific knockout mouse lines N‐KO & C‐KO were created. After 14 day Ang II infusion, BP was 27 mm Hg (p<0.0001) higher in N‐KO vs. WT/C‐KO. N‐KO kidney Ang II levels were 1.5‐fold those of WT/C‐KO (p<0.015). Peritoneal macrophage cytokine expression in response to Ang II, measured as percentage of F4/80+/TNFαhigh cells vs total F4/80+, was 40.3% ± 4.7% for N‐KO and 19.8% ± 2.8% for WT (p<0.005). In response to LPS, N‐KO macrophages made 4‐fold the TNFα as C‐KO/WT cells (p<0.001). AcSDKP, a substrate specifically cleaved by the N‐domain, is made by the enzyme prolyl‐oligopeptidase (POP). Daily injections of the POP inhibitor S‐17092 were given to N‐KO and WT mice. This reduced the LPS‐mediated N‐KO macrophage production of TNFα to that of cells from WT. The hypertensive response to Ang II was also tested in N‐KO and WT mice treated with daily S‐17092. POP inhibition had no effect on the BP of WT, but reduced the BP of N‐KO to that of WT (p<0.01). Furthermore, FACS analysis of peritoneal macrophages showed a decreased percentage of F4/80+/TNFαhigh cells in N‐KO mice treated with daily S‐17092. The lack of N domain activity in ACE exhibited a pro‐inflammatory role in experimental hypertension via cytokine regulation, accumulation of renal Ang II, and increased BP response. Inhibition of POP eliminated the differences in macrophage TNFα expression and blood pressure response between N‐KO and WT mice.

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells.

Chemical structure and immunobiological activity of lipid A from Serratia marcescens LPS

The chemical structure and immunobiological activities of Serratia marcescens lipid A, an active centre of LPS, were investigated. LPS preparations of S. marcescens were extracted using a hot phenol/water method, after which purified lipid A specimens were prepared by weak acid hydrolysis, followed by normal phase and gel filtration chromatographic separation. The lipid A structure was determined by MS to be a diglucosamine backbone with diphosphates and five C(14) normal chain acyl groups, including two acyloxyacyl groups at the 2 and 3 positions of the non-reducing side. S. marcescens lipid A and Escherichia coli-type synthetic lipid A (compound 506) exhibited definite reactivity in Limulus amoebocyte lysate assays. The lethal toxicity of S. marcescens lipid A was nearly comparable to that of compound 506, and both induced nuclear factor-kappaB activation in murine cells via Toll-like receptor (TLR)4/MD-2 but not TLR2, as well as various inflammatory cytokines in peritoneal macrophages of C3H/HeN mice but not C3H/HeJ mice. Furthermore, S. marcescens lipid A induced nearly the same amounts of tumour necrosis factor alpha, interleukin-6, and nitric oxide production by the murine alveolar macrophage cell line MH-S as compared with compound 506. These results indicate that S. marcescens possesses a penta-acylated lipid A, which is nearly identical to E. coli lipid A in regard to biological activities, while it also may be a crucial virulence factor of the bacterium.

Ganoderma tsugae in vivo modulates Th1/Th2 and macrophage responses in an allergic murine model

We have reported that Ganoderma tsugae supplementation alleviates bronchoalveolar inflammation in an airway sensitization and challenge model with female BALB/c mice. However, the effects of G. tsugae supplementation in vivo on serum antibody levels, splenocyte and peritoneal microphage immune responses have not yet been determined. In this study, serum antibody levels, cytokines and splenocyte chemical mediators and peritoneal macrophage cultures from ovalbumin (OVA)-sensitized and -challenged mice were examined after continuously consuming G. tsugae supplementation diets for 5 weeks. The results showed that OVA sensitization and challenge significantly ( P < 0.05) decreased the spontaneous production of IL-2 (Th1) cytokine, but significantly ( P < 0.05) increased spontaneous and OVA-stimulated IL-4 (Th2) production in splenocyte cultures from experimental mice. OVA administration significantly decreased both spontaneous and LPS/IFN-γ-stimulated IL-1β and IL-6 levels in peritoneal macrophage cultures from experimental mice. However, dietary supplementation with G. tsugae significantly increased spontaneous IL-2 level, but slightly decreased spontaneous IL-4 level in cultured splenocyte supernatants in the experimental groups. G. tsugae supplementation enhanced pro-inflammatory cytokines IL-1β and IL-6 production in cultured peritoneal macrophages. However, the nitric oxide level from cultured peritoneal macrophages and serum OVA-specific IgE and IgG 2a antibody levels was not significantly affected. These results suggest that OVA sensitization and challenge induced a Th2-skewed splenocyte response and decreased peritoneal macrophage cytokine secretion. G. tsugae supplementation in vivo modulated the Th1/Th2 balance and enhanced macrophage immune responses. However, the supplementation diet could not fully reverse the Th2-skewed responses to level of Th1-skewed responses.

Different ratios of eicosapentaenoic and docosahexaenoic omega-3 fatty acids in commercial fish oils differentially alter pro-inflammatory cytokines in peritoneal macrophages from C57BL/6 female mice

The use of fish oil (FO) as a dietary supplement to prevent or reduce the severity of cardiovascular diseases and autoimmune disorders such as rheumatoid arthritis is receiving much attention. Several recent reports indicate that eating fish often or the use of small doses of FO capsules appears to have benefits against cardiovascular diseases. We have reported in the past that diets enriched with FO protect against renal diseases and prolong the life span of autoimmune-prone mice compared to corn oil (CO) diets. However, the optimum ratio of eicosapentaenoic acid (EPA) to docosahexaenoic acid (DHA) in commercially available FOs to reduce the production of various pro-inflammatory cytokines has not been well established. We, therefore, obtained deodorized FO from three sources containing different EPA/DHA contents, fed them to C57BL/6 mice for 8 weeks in a 10% (vol/wt) diet (oil A, 11/10; oil B, 14/9; oil C, 23/14) and compared them with (10%) CO-fed mice as control. TNF-α, IL-6 and IL-1β were measured by enzyme-linked immunosorbent assay in thioglycollate-induced macrophages, 8 and 24 h after lipopolysaccharide treatment. The results showed a significant decrease in TNF-α after only 8 h in oil C. After 24 h, TNF-α, IL-6 and IL-1β levels decreased only in mice fed oil C, although nonsignificant decreases were seen in mice fed oil A compared to mice fed CO. The antioxidant enzymes, catalase and glutathione transferase, were higher in kidneys of mice fed oil C compared to mice fed CO. The study suggests that anti-inflammatory activity may vary among different sources of FO due to variations in EPA/DHA content.

CO 2 pneumoperitoneum attenuates IL-6 production in peritoneal macrophages

The accelerated recovery of patients who undergo laparoscopic surgery is often attributed to the less traumatic approach. However, a growing body of evidence suggests that the capnoperitoneum specifically has antiinflammatory effects. While the cellular target of CO 2 has yet to be identified, the peritoneal macrophage is a prime target because of its major role in the inflammatory response. We hypothesized that the antiinflammatory effect of capnoperitoneum may be mediated via inhibition of peritoneal macrophage cytokine production. Male Sprague-Dawley rats were randomized to CO 2 pneumoperitoneum, anesthesia only, bacterial endotoxin (LPS) only, or saline ( n = 4 for all groups). Rats underwent 30 min of pneumoperitoneum or anesthesia followed immediately by intravenous injection of LPS. Controls received either LPS or saline without a prolonged exposure to anesthesia. One hour following LPS injection, peritoneal macrophages were harvested by peritoneal lavage and incubated for 3 h at 37°C in complete medium. IL-6 was measured during the ensuing 3 h using a standard ELISA kit. Values were normalized to cell number using the MTT uptake method. IL-6 production was significantly attenuated in macrophages derived from rats which had undergone capnoperitoneum and resembled the basal production found in macrophages harvested from saline controls ( P < 0.013 versus LPS and P < 0.03 versus Isoflurane). Macrophages harvested from rats having undergone anesthesia alone produced IL-6 in similar quantities to macrophages from LPS-treated controls. All samples were measured in duplicate and normalized to viable cell number. These results confirm our hypothesis and demonstrate that the advantages of laparoscopic surgery are in part mediated by the peritoneal macrophage. Capnoperitoneum when applied prior to an inflammatory insult in vivo significantly attenuates cytokine production.

Automated Peritoneal Dialysis Symposium: Host Defenses in APD

The incidence of peritonitis in peritoneal dialysis (PD) has further decreased after the introduction of automated peritoneal dialysis (APD) into clinical routine. Technical advances such as a reduction in the number of connections, more motivated patients, and improved host defense mechanisms in APD have been described. We found that the long daytime interval without dialysate exchanges leads to a higher absolute number of peritoneal macrophages which are capable of an improved first‐line host defense. This is demonstrated by a higher release of cytokines of peritoneal macrophages after stimulation with lipopolysaccharides. The dry “day” in nightly intermittent PD seems to have no relevant additional positive effects on peritoneal host defense compared to continuous cyclic peritoneal dialysis. The regeneration of peritoneal cell populations induced by intervals without PD remains relevant even in times when more “biocompatible” PD solutions become available.

Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75-mg morphine slow-release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 microg/mL) plus interferon-gamma (IFN-gamma: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase-polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme-linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)-12 p40 and tumor necrosis factor alpha (TNF-alpha) compared with controls, whereas IL-10 levels were unchanged by drug treatment. ELISA data showed that both IL-12 p40 and p70 were increased by morphine. The enhancement of IL-12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram-negative and other enteric bacteria. The enhancement of IL-12 by morphine might be related to morphine-induced sepsis.

Alteration of cytokine production in follicular cystic ovaries induced in mice by neonatal estradiol injection.

Neonatal estradiol injections in mice lead to follicular cystic ovaries that are similar to ovaries in patients with polycystic ovarian syndrome (PCOS). The present study examined ovarian cytokine production following neonatal estradiol injection. Female (C3H,HeJ x 129/HeJ)F1 mice were injected daily with 20 microg 17beta-estradiol from 0-3 days postpartum. At intervals, animals were sacrificed to determine ovarian architecture, circulating levels of estradiol, ovarian and peritoneal macrophage cytokine production, and ovarian P450 aromatase enzyme mRNA levels. Similar to PCOS, our results show that neonatally estradiol-injected mice have lower levels of circulating estrogen that are correlated with decreased mRNA levels of P450 aromatase enzyme. Our data also show that follicular cystic ovaries have increased tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production. This increase in TNF-alpha and IL-6 production is also observed in peritoneal macrophages of estradiol-injected mice. The present study showed that neonatal estrogen injection in mice has an overall systemic effect on cytokine production. We speculate that increased cytokine production may alter certain important steps in follicular maturation, ultimately contributing to ovarian dysfunction.

Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses

Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.