小檗碱与育亨宾对脓毒症小鼠脾细胞凋亡的影响及其机制研究

 目的:探讨小檗碱与育亨宾对脓毒症小鼠脾细胞凋亡的影响及其作用机制。方法: 采用盲肠结扎穿孔（CLP）构建小鼠脓毒症模型,分为假手术（sham）组、CLP组、CLP+小檗碱组、CLP+育亨宾组、CLP+小檗碱与育亨宾合剂组。CLP术后2 h灌胃给予相应药物,20 h后取脾脏,用TUNEL和流式细胞术检测小鼠脾细胞凋亡,酶荧光法检测caspase-3、caspase-8和caspase-9的活性变化,Western blotting检测凋亡相关蛋白Fas、Bim、Bcl-2和Bax的表达。结果: (1) CLP组脾脏TUNEL阳性细胞百分率显著高于sham组（P＜0.05）,CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组凋亡细胞百分率显著低于CLP组（P＜0.05）。(2) 流式细胞仪检测显示CLP组凋亡的脾细胞及T淋巴细胞明显多于sham组(P＜0.05),CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组凋亡的脾细胞及T淋巴细胞明显少于CLP组 (P＜0.05) 。(3) CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组脾细胞caspase-3、caspase-8、caspase-9的活性均低于CLP组(P＜0.05);而CLP+小檗碱组脾细胞caspase-9活性也低于CLP组 (P＜0.05)。(4) CLP+育亨宾与小檗碱合剂组胞浆Fas、Bim、Bax表达均低于CLP组,CLP+育亨宾组胞浆Fas表达低于CLP组,CLP+小檗碱治疗组胞浆Bim、线粒体Bax表达均低于CLP组。结论: (1) 小檗碱与育亨宾合用可通过阻断内、外源性凋亡途径抑制脓毒症小鼠脾细胞凋亡,特别是T淋巴细胞凋亡。(2) 育亨宾主要通过抑制Fas的表达、进而阻断内、外源性凋亡途径减少脓毒症诱导的脾细胞凋亡。(3) 小檗碱可抑制脓毒症小鼠脾细胞线粒体凋亡途径,但对脓毒症小鼠脾细胞凋亡的抑制作用并不明显。     

CD8+ T cells promote inflammation and apoptosis in the liver after sepsis: role of Fas-FasL

Although studies blocking the Fas pathway indicate it can decrease organ damage while improving septic (cecal ligation and puncture, CLP) mouse survival, little is known about how Fas-Fas ligand (FasL) interactions mediate this protection at the tissue level. Here, we report that although Fas expression on splenocytes and hepatocytes is up-regulated by CLP and is inhibited by in vivo short interfering RNA, FasL as well as the frequency of CD8(+) T cells are differentially altered by sepsis in the spleen (no change in FasL, decreased percentage of CD8(+) and CD4(+) T cells) versus the liver (increased FasL expression on CD8(+) T cells and increase in percentage/number). Adoptive transfer of CLP FasL(+/+) versus FasL(-/-) mouse liver CD8(+) T cells to severe combined immunodeficient or RAG1(-/-) recipient mice indicated that these cells could induce inflammation. The FasL-mediated cytotoxic capacity of these septic mouse liver CD8(+) T cells was shown by their ability to damage directly cultured hepatocytes. Finally, although CD8(-/-) mice exhibited a reduction in both CLP-induced liver active caspase-3 staining and blood interleukin-6 levels, only FasL(-/-) (but not CD8(-/-)) protected the septic mouse spleen from increasing apoptosis. Thus, although truncating Fas-FasL signaling ameliorates many untoward effects of sepsis, the pathological mode of action is distinct at the tissue level.     

Are alterations of lymphocyte subpopulations in polymicrobial sepsis and DHEA treatment mediated by the tumour necrosis factor (TNF)‐α receptor (TNF‐RI)? A study in TNF‐RI (TNF‐RI–/–) knock‐out rodents

Sepsis is associated with depression of T cell-dependent immune reactivity with proinflammatory cytokines, such as tumour necrosis factor (TNF)-alpha, playing an important role. Recent investigations describe an association between these immunological alterations and disturbances of the endocrine system, related most frequently to sex steroid hormones. Dehydroepiandrosterone (DHEA), one of the most abundant adrenal sex steroid precursors, seems to have a protective immunological effect towards septic insults. In this study, both the role of TNF-receptor I (RI) and possible interactions in the protective role of DHEA were investigated in a murine model of polymicrobial sepsis. Polymicrobial sepsis was induced by caecal ligation and puncture (CLP) in a murine model. The effects of DHEA on survival, clinical parameters and cellular immunity (T lymphocytes and natural killer (NK) cells) were investigated. CLP was performed in genetically modified TNF-RI knock-out (TNF-RI(-/-)) and genetically unmodified (wild-type, WT) mice. DHEA application was associated with a decrease in the mortality rate in WT animals. A mortality rate of 91.7% was observed in TNF-RI(-/-) mice after CLP. This mortality rate was reduced to 37.5% by the application of DHEA. In sham-operated TNF-RI(-/-) animals, a significantly higher proportion of NK cells within the lymphocyte population was measured compared with the corresponding WT group. After CLP, a significant increase in the percentage cell count of NK cells was recorded in WT mice. Overall, following DHEA application in WT mice, an alteration in the cellular immune response was characterized by a reduction in the percentage counts of CD4(+), CD8(+) and NK cells. In the group of TNF-RI(-/-) mice treated with DHEA, no increase in the percentage cell count of NK cells was observed after CLP. No data for cell analysis were available from the CLP-TNF-RI(-/-) mice treated with saline, due to the high mortality rate in these animals. DHEA reduces the complications of sepsis in a TNF-RI-independent manner. Our study suggests that NK cells are involved in the protective mechanism of DHEA in WT mice. It would therefore seem that DHEA represents a feasible alternative therapy for the dysregulated immune system in sepsis.     

Reduced Expression of SARM in Mouse Spleen during Polymicrobial Sepsis

Objective Immune dysfunction, including prominent apoptosis of immune cells and decreased functioning of the remaining immune cells, plays a central role in the pathogenesis of sepsis. Sterile α and HEAT/armadillo motif-containing protein (SARM) is implicated in the regulation of immune cell apoptosis. This study aimed to elucidate SARM contributes to sepsis-induced immune cell death and immunosuppression. Methods A mouse model of polymicrobial sepsis was generated by cecum ligation and puncture (CLP). SARM gene and protein expression, caspase 3 cleavage and intracellular ATP production were measured in the mouse spleens. Results CLP-induced polymicrobial sepsis specifically attenuated both the gene and protein expression of SARM in the spleens. Moreover, the attenuation of SARM expression synchronized with splenocyte apoptosis, as evidenced by increased caspase 3 cleavage and ATP depletion. Conclusions These findings suggest that SARM is a potential regulator of sepsis-induced splenocyte apoptosis.     

Immunosuppressive activity of florfenicol on the immune responses in mice

Florfenicol is a new type of broad-spectrum antibacterial that has been used in veterinary clinics. It shows immunosuppressive activity on the immune responses to ovalbumin (OVA) in mice. In the present study, florfenicol suppressed lipopolysaccharide (LPS)-stimulated splenocyte proliferation in a concentration-dependent manner in vitro and in vivo. BALB/c mice were immunized subcutaneously with OVA on days 1 and 4. Following the second immunization, mice were treated with a single daily oral dose of florfenicol (50, 100, and 200 mg/kg) for 10 consecutive days. On day 14, blood samples were collected to analyze OVA-specific IgG, IgG1, and IgG2b antibodies, and splenocytes were harvested to assess lymphocyte proliferation, CD3(+) T and CD19(+) B lymphocyte subsets. The results presented here demonstrate that florfenicol not only significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation but also decreased the percentage of CD19(+) B cells in a dose-dependent manner and suppressed CD3(+) T cell at high doses. Moreover, OVA-specific IgG, IgG1 and IgG2b titers in OVA-immunized mice were reduced by florfenicol. These results suggest that florfenicol could suppress humoral and cellular immune responses in mice.     

Inhibition of Fas/Fas ligand signaling improves septic survival: differential effects on macrophage apoptotic and functional capacity

Signaling through the Fas/Fas ligand (FasL) pathway plays a central role in immune-cell response and function; however, under certain pathological conditions such as sepsis, it may contribute to the animal's or patient's morbidity and mortality. To determine the contribution of FasL to mortality, we conducted survival studies by blocking Fas/FasL with Fas receptor fusion protein (FasFP) in vivo. C3H/HeN mice received FasFP or the saline vehicle (veh) immediately (0 h) or delayed (12 h), after sepsis induced by cecal ligation and puncture (CLP). Subsequently, we examined the effect of FasFP treatment (12 h post-CLP) on macrophage apoptosis and functional capacities. Peritoneal and splenic macrophages and Kupffer cells from sham-veh-, CLP-veh-, sham-FasFP-, or CLP-FasFP-treated mice were harvested 24 h after CLP and stimulated with lipopolysaccharide (LPS) for 24 h. The results indicate that only delayed (12 h) but not 0 h administration of FasFP demonstrated a significant increase in survival. The ability of all macrophage populations to release interleukin (IL)-6 was significantly depressed, and IL-10 release was augmented after CLP. FasFP treatment attenuated the increased IL-10 release in Kupffer cells. However, althogh enhanced susceptibility to LPS-induced apoptosis could be suppressed in CLP mouse Kupffer cells by FasFP, FasFP did not change the peritoneal or splenic macrophage response. Furthermore, FasFP attenuated the elevated plasma levels of liver enzymes after sepsis. These data indicate that in vivo inhibition of Fas/FasL signaling has tissue-specific effects on the induction of macrophage apoptosis, functional changes, and liver damage, which may contribute to the host's ability to ward off a septic challenge.     

TRH receptor on immune cells:In vitro andin Vivo stimulation of human lymphocyte and rat splenocyte DNA synthesis by TRH

This work examined whether (1) immune cells express thyrotrophin releasing hormone (TRH) receptor mRNA and (2) TRH modulates lymphocyte activation. By Northern blot of RNA extracted from human peripheral blood mononuclear cells (PBMC) and rat splenocytes, a single TRH receptor mRNA band of about 3.8 kb (identical to that obtained from pituitary cells) was obtained, under both basal and stimulated conditions. A significant increase in DNA synthesis was observed in phytohemagglutinin-stimulated PBMC and concanavalin A (Con A) stimulated splenocytes when TRH (10^–6 M-10^–12 M) was added. After 5, 30, 60, 180 min and 24 h of TRH administrationin vivo, a significant increase in the rat splenocyte proliferative response to Con A was observed.In vivo administration of anti-rat TSH antibody (1/1000) blocked the increase observed after 30 min of TRH administration on the Con A stimulated splenocyte response. TRH possess immunostimulatory functions directly via its receptor and indirectly via release of other immunostimulatory factors such as thyrotrophin.     

胸腺五肽对鼠免疫功能的影响

本文研究了合成胸腺五肽(TP-5)对正常小鼠和老龄大鼠免疫功能的影响。TP-50.5,1,2,5mg/kg ip×3d可明显增强脾淋巴细胞的增殖反应,选择性地抑制脾细胞溶血空斑数(PFC),TP-52,5mg/kg可显著增加ICR小鼠胸腺的重量,用5mg/kg TP-5可对抗50mg/kg环孢霉素A(CysA)对淋巴细胞增殖的抑制作用。体外脾细胞培养中,TP-51,5,10,20μg/ml与ConA协同作用能明显增强小鼠脾淋巴细胞的增殖,同样,对老龄大鼠TP-5具有相同的刺激作用。     

Immunosuppressive Activity of Florfenicol on the Immune Responses in Mice

Florfenicol is a new type of broad-spectrum antibacterial that has been used in veterinary clinics. It shows immunosuppressive activity on the immune responses to ovalbumin (OVA) in mice. In the present study, florfenicol suppressed lipopolysaccharide (LPS)-stimulated splenocyte proliferation in a concentration-dependent manner in vitro and in vivo. BALB/c mice were immunized subcutaneously with OVA on days 1 and 4. Following the second immunization, mice were treated with a single daily oral dose of florfenicol (50, 100, and 200 mg/kg) for 10 consecutive days. On day 14, blood samples were collected to analyze OVA-specific IgG, IgG1, and IgG2b antibodies, and splenocytes were harvested to assess lymphocyte proliferation, CD3⁺ T and CD19⁺ B lymphocyte subsets. The results presented here demonstrate that florfenicol not only significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation but also decreased the percentage of CD19⁺ B cells in a dose-dependent manner and suppressed CD3⁺ T cell at high doses. Moreover, OVA-specific IgG, IgG1 and IgG2b titers in OVA-immunized mice were reduced by florfenicol. These results suggest that florfenicol could suppress humoral and cellular immune responses in mice.     

Farnesyltransferase inhibitor FTI‐277 inhibits PD‐L1 expression on septic spleen lymphocytes and promotes spleen lymphocyte activation

Farnesyltransferase inhibitors have been tested in clinical trials for the treatment of tumours. In sepsis, the binding of programmed death 1 (PD‐1) to programmed death ligand 1 (PD‐L1) promotes lymphocyte apoptosis and decreases cytokine expression, thus affecting survival rates. The PD‐1/PD‐L1 pathway plays an important role in chronic viral infection, bacterial infection and sepsis. However, the precise immunosuppressive and anti‐inflammatory functions of this pathway remain poorly understood. In our previous study, the induction of sepsis by caecal ligation and puncture (CLP) resulted in increased farnesyltransferase activity and farnesylated protein levels in the spleen relative to sham treatment. However, the effect of inhibition of farnesyltransferase activity on overall survival rates in patients with sepsis and the specific signalling pathway involved remain to be investigated. In this study, mice with CLP‐induced sepsis were treated with farnesyltransferase inhibitor (FTI‐277), and PD‐L1 expression on septic spleen lymphocytes was examined. Flow cytometric analysis revealed that PD‐L1 is expressed constitutively on lymphocytes and that PD‐L1 protein expression was up‐regulated strongly following CLP. FTI‐277 down‐regulated PD‐L1 mRNA and protein expression on septic spleen lymphocytes in a dose‐dependent manner. This effect was associated closely with nuclear factor kappa B (NF‐κB). In addition, the significant damping effect of FTI‐277 on the PD‐L1 signal promoted interferon (IFN)‐γ secretion, interleukin (IL)‐2 production and splenocyte proliferation in response to anti‐CD3⁺CD28⁺ antibodies in mice. Furthermore, FTI‐277 reduced spleen lymphocyte apoptosis in septic mice. Therefore, FTI‐277 regulates spleen lymphocyte activity via the PD‐L1 signalling pathway, with significant anti‐inflammatory effects attributable to suppression of the NF‐κB pathway. Farnesyltransferase represents a valuable therapeutic target for the treatment of sepsis.     

Effects of simvastatin on the function of splenic CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells in sepsis mice

Simvastatin may be beneficial for treating sepsis due to its immune-regulating properties, although the mechanisms remain elusive. Herein, we hypothesized simvastatin may attenuate T cell dysfunction induced by sepsis. To test this hypothesis, we used a model based on cecal ligation and puncture (CLP) to induce sepsis in mice. Male C57BL/6 mice were pre-treated with simvastatin (0.2 μg/g of body weight) before CLP. The expression of B and T lymphocyte attenuator (BTLA) on splenic CD4⁺ T cells and T cell apoptosis, CD4⁺ and CD8⁺ T cells were quantified by flow cytometry. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. Formation of TNF-α and interleukin 10 (IL-10) in the spleen and plasma levels of presepsin, IL-1β, and IL-6 were determined using enzyme-linked immunosorbent assay. Simvastatin markedly inhibited the reduction in cytokine secretion from lipopolysaccharide (LPS)-stimulated splenocytes. Simvastatin-treated mice had significantly decreased the percentages of negative costimulatory receptor BTLA on CD4 T cell expression. Simvastatin markedly reduced T cell apoptosis through downregulating the Fas/FasL expression and decrease the percentage of caspase-3 activity in spleen tissue. There was significantly less depletion of splenic CD4⁺ and CD8⁺ T cells in simvastatin-treated mice. Simvastatin reduced plasma levels of presepsin, IL-1β, and IL-6. Simvastatin can be a powerful regulator of immune function under sepsis conditions by improving T cell function in sepsis.     

Effect of Systemic Candidiasis on Blastogenesis of Lymphocytes from Germfree and Conventional Rats

Germfree and conventional rats were challenged (intravenously) with Candida albicans and sacrificed at various times after infection, and their spleen cells were harvested to examine the effect of disseminated candidiasis on in vitro lymphocyte hypersensitivity to Candida antigens (CA). Results showed that conventional rat splenocytes, initially responsive in vitro to stimulation by CA, manifested a depression in CA-specific responsiveness after challenge with viable C. albicans (days 3 to 6 postchallenge). In addition, the latter splenocyte response to phytohemagglutinin (PHA) and concanavalin A (ConA) was suppressed by 3 to 6 days after challenge with Candida. In contrast to conventional rats, the response of germfree rat splenocytes to CA was insignificant before challenge with C. albicans, and it was increased at 9 days after infection. The response of uninfected germfree rat splenocytes to PHA and ConA was significantly lower than that of unchallenged conventional rats. Challenge with viable C. albicans did not result in a suppression of gnotobiotic rat splenocyte responses to PHA and ConA, but rather, the disseminated infection resulted in as much as fivefold increases in PHA or ConA-induced blastogenesis. These findings suggest that disseminated candidiasis is capable of suppressing blastogenesis in immunologically mature conventional rats and of improving lymphocyte blastogenesis from immunologically immature germfree rats.     

Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin.

The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-alpha was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1 beta was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-alpha with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-alpha with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N(G)-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.     

Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis

Earlier work from our laboratory has suggested a role for the neuropeptide substance P (SP) in inducing lung injury in sepsis. In that study, mice lacking the preprotachykinin-A gene, which encodes for SP, were protected against lung injury in sepsis. To further substantiate the role of SP in sepsis and to study its mechanism, we have evaluated the effect of SR140333, a SP receptor antagonist, on lung injury in sepsis, which was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham-operated animals received the same surgical procedure, except CLP. Vehicle or SR140333 (1 mg/kg, s.c.) was administered to CLP mice 30 min before or 1 h after the CLP. Eight hours after surgery, lung tissue was collected and analyzed for myeloperoxidase (MPO) activity, chemokines, cytokines, and adhesion molecules. The CLP procedure alone caused a significant increase in the lung levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, E- and P-selectin, and MPO activity when compared with sham-operated mice. SR140333 injected 30 min before or 1 h after CLP significantly attenuated the increased lung MPO activity and levels of MIP-2, MCP-1, IL-1beta, IL-6, ICAM-1, and E- and P-selectin compared with CLP-operated mice injected with the vehicle. Histological evaluation of the lung sections further supported the beneficial effect of SR140333 on lung inflammation. Therefore, SP receptor antagonism can be a potential therapeutic target in polymicrobial sepsis, and this effect is brought about via reduction in leukocyte recruitment.     

Gammadelta T cells mitigate the organ injury and mortality of sepsis

Sepsis is a difficult condition to treat and is associated with a high mortality rate. Sepsis is known to cause a marked depletion of lymphocytes, although the function of different lymphocyte subsets in the response to sepsis is unclear. gammadelta T cells are found largely in epithelial-rich tissues, and previous studies of gammadelta T cells in models of sepsis have yielded divergent results. In the present study, we examined the function of gammadelta T cells during sepsis in mice using cecal ligation and puncture (CLP). Mice deficient in gammadelta T cells had decreased survival times and increased tissue damage after CLP compared with wild-type mice. Furthermore, bacterial load was increased in gammadelta T cell-deficient mice, yet antibiotic treatment did not change mortality. Additionally, we found that recruitment of neutrophils and myeloid suppressor cells to the site of infection was diminished in gammadelta T cell-deficient mice. Finally, we found that circulating levels of IFN-gamma were increased, and systemic levels of IL-10 were decreased in gammadelta T cell-deficient mice after CLP compared with wild-type mice. gammadelta T cell-deficient mice also had increased intestinal permeability after CLP compared with wild-type mice. Neutralization of IFN-gamma abrogated the increase in intestinal permeability in gammadelta T cell-deficient mice. The intestines taken from gammadelta T cell-deficient mice had decreased myeloperoxidase yet had increased tissue damage as compared with wild-type mice. Collectively, our data suggest that gammadelta T cells modulate the response to sepsis and may be a potential therapeutic target.     

Beta-arrestin 2 negatively regulates sepsis-induced inflammation

β-arrestins 1 and 2 are ubiquitously expressed proteins that alter signalling by G-protein-coupled receptors. β-arrestin 2 plays an important role as a signalling adaptor and scaffold in regulating cellular inflammatory responses. We hypothesized that β-arrestin 2 is a critical modulator of inflammatory response in experimental sepsis. β-arrestin 2(−/−) and wild-type (WT) mice were subjected to caecal ligation and puncture (CLP). The survival rate was significantly decreased (P < 0·05) in β-arrestin 2(−/−) mice (13% survival) compared with WT mice (53% survival). A second group of mice were killed 18 hr after CLP for blood, peritoneal lavage and tissue sample collection. CLP-induced plasma interleukin (IL)-6 was significantly increased 25 ± 12 fold and caecal myeloperoxidase (MPO) activity was increased 2·4 ± 0·3 fold in β-arrestin 2(−/−) compared with WT mice. β-arrestin 2(−/−) mice exhibited more severe lung damage and higher bacterial loads compared with WT mice post CLP challenge as measured by histopathology and colony-forming unit count. In subsequent experiments, splenocytes, peritoneal macrophages and bone marrow-derived macrophages (BMDMs) were isolated and cultured from β-arrestin 2(−/−) and WT mice and stimulated in vitro with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-α, IL-6 and IL-10 production induced by LPS was significantly augmented (2·2 ± 0·2 fold, 1·8 ± 0·1 fold, and 2·2 ± 0·4 fold, respectively; P < 0·05) in splenocytes from β-arrestin 2(−/−) mice compared with WT mice. The splenocyte response was different from that of peritoneal macrophages or BMDMs, which exhibited no difference in TNF-α and IL-6 production upon LPS stimulation between WT and β-arrestin 2(−/−) mice. Our data demonstrate that β-arrestin 2 functions to negatively regulate the inflammatory response in polymicrobial sepsis.     

Interleukin 3 activity in tumor-bearing hosts: decreased splenocyte production of and responsiveness to IL 3

A kinetic study assessing the relationship between tumor growth and the ability of BALB/c mouse splenocytes to produce Interleukin 3 (IL 3) indicated a concomitant decrease in IL 3 activity with tumor growth. Tumor-bearing host (TBH) splenocytes produced 600 pmoles/hr/10(8) cells of IL 3 activity at Day 0 but only 62 pmoles/hr/10(8) cells by Day 28 post tumor cell inoculation. Nylon wool fractionation (to remove adherent suppressor cells) did not restore IL 3 activity. Addition of purified IL 3 to mitogen proliferation assays showed that IL 3 alone was mitogenic for normal host but not TBH splenocytes. In concert with concanavalin A (Con A) and phytohemagglutinin, IL 3 augmented in vitro normal host splenocyte responsiveness but significantly further suppressed it in the TBH. An absorption assay indicated that fresh cells had acceptors to remove IL 3 from supernatants. Con A-induced normal or TBH blast cells lost their ability to absorb IL 3. Intravenous inoculation of purified IL 3 into normal and TBH resulted in further suppression of TBH splenocyte mitogen-induced blastogenesis. The exacerbation of TBH spleen cell reactivity by IL 3 may be due to a tumor-induced feedback inhibition mechanism further suppressing cellular differentiation critical to cytotoxic T lymphocyte maturation.     

Gene Dosage-Dependent Negative Regulatory Role of β-Arrestin-2 in Polymicrobial Infection-Induced Inflammation

β-arrestin-2 (β-arr2) is a scaffolding protein of the arrestin family with a wide variety of cellular functions. Recent studies have demonstrated differential roles for β-arr2 in inflammation following endotoxemia and cecal ligation and puncture (CLP) models of sepsis. Because CLP-induced inflammation involves response to fecal contents and necrotic cecum in addition to microbial challenge, in this study, we examined the role of β-arr2 in an exclusively polymicrobial infection (PMI) model. In addition, we examined the role of gene dosage of β-arr2 in polymicrobial sepsis. Our studies demonstrate that β-arr2 is a negative regulator of systemic inflammation in response to polymicrobial infection and that one allele is sufficient for this process. Our results further reveal that loss of β-arr2 leads to increased neutrophil sequestration and overt inflammation specifically in the lungs following polymicrobial infection. Consistent with this, specific NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways were differentially activated in the β-arr2 knockout (KO) mice lungs compared to the wild type (WT) following PMI. Associated with enhanced inflammation in the KO mice, PMI-induced mortality was also significantly higher in KO mice than in WT mice. To understand the differential role of β-arr2 in different sepsis models, we used cell culture systems to evaluate inflammatory cytokine production following endotoxin and polymicrobial stimulation. Our results demonstrate cell-type- as well as stimulus-specific roles for β-arr2 in inflammation. Taken together, our results reveal a negative regulatory role for β-arr2 in polymicrobial infection-induced inflammation and further demonstrate that one allele of β-arr2 is sufficient to mediate most of these effects.     

Down-regulation of murine lymphocyte responsiveness to mitogens after treatment with antigens ofEntamoeba histolytica

Injection of mice with pathogenicEntamoeba histolytica (strain HM1-IMSS) antigens resulted in a decreased capacity of splenocytes to respond to mitogeninduced blastogenesis following a challenge with concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS), whereas no inhibition was observed in mice that had previously been injected with equivalent amounts of non-pathogenicE. histolytica-like Laredo antigens. Depletion of adherent cells in the splenocyte preparation indicated that these cells were not a major contributor to the observed immunosuppression. Quantification of splenic T-lymphocyte subsets demonstrated a significant decrease in Thy-1⁺ and Lyt-1⁺ cells, but Lyt-2⁺ cells were not affected. Splenocytes treated with pathogenic amoebic antigens in vitro affected the capacity of these cells to respond optimally to Con A-and LPS-induced blastogenesis but not to that induced by PHA. These findings demonstrate that amoebic antigens affect lymphocyte function and may be important co-factors in the immunoregulation and pathogenesis of amoebiasis.