M1和M2型巨噬细胞表型的比较分析

通过对M1和M2型巨噬细胞表型相关指标的比较分析,评价各鉴定巨噬细胞类型的表型指标及其意义。按常规方法以IFN-γ及LPS将骨髓来源巨噬细胞诱导成M1型巨噬细胞,以IL-4诱导出M2型巨噬细胞。分别以RT-PCR和酶活性定量方法检测精氨酸代谢相关酶的表达和活性;以ELISA检测IL-12和IL-10的分泌;以FACS检测巨噬细胞膜分子的表达。结果显示:M1型巨噬细胞诱导性一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达和活性水平较未刺激组明显升高,IL-12产生显著增加,CD16/32表达上调;而M2型巨噬细胞I型精氨酸酶(arginase 1,Arg-1)的表达水平和酶活性较未刺激巨噬细胞显著提高,IL-10分泌轻度增加,并且表达高水平的CD206和DECTIN-1。表型比较分析结果表明,iN-OS表达和活性、IL-12的分泌和膜蛋白CD16/32可用于鉴定M1型巨噬细胞,而Arg-1、CD206和DECTIN-1是鉴定M2型巨噬细胞较为理想的表型指标。     

Leishmania mexicana promastigotes inhibit macrophage IL-12 production via TLR-4 dependent COX-2, iNOS and arginase-1 expression

The effects of Leishmania mexicana metacyclic promastigotes upon MAP kinase signalling in mouse bone marrow macrophages and subsequent expression of the disease regulatory proteins iNOS and COX-2 were studied. At a ratio of 5:1, promastigotes caused a marked increase in phosphorylation of the three major MAP kinases, ERK, p38 and JNK. MAP kinase signalling was substantially reduced in TLR-4^−/− but not TLR-2^−/− deficient macrophages and completely abolished in double TLR-2/4^−/− macrophages. A similar outcome was observed using cysteine peptidase B deficient amastigotes. Furthermore, whilst promastigotes had no independent effect on iNOS or COX-2 expression, they prolonged the induction of these proteins stimulated by LPS and enhanced PGE₂ and NO production. Induction of COX-2 and iNOS was also TLR-4 dependent. Blockade of either PGE₂ or NO production with indomethacin or l-NAME reversed promastigote inhibition of LPS induced IL-12 production. Promastigotes also increased macrophage arginase-1 expression and enhanced arginase activity, both of which were substantially reduced in TLR-4 but not TLR-2 deficient macrophages. Surprisingly, arginase inhibition by Nor-NOHA also caused a reversal of promastigote mediated inhibition of macrophage IL-12 production. These data demonstrate for the first time the role of TLR-4 in mediating the effects of L. mexicana promastigotes on MAP kinase activation, up-regulation of COX-2, iNOS as well as arginase-1 expression in macrophages and further shows that PGE₂, NO and arginase activity all contribute substantially to the inhibition of host cell IL-12 production.     

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.     

Interleukin-10 inhibits IgE-mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes

Human keratinocytes (HK) generate nitric oxide (NO) and proinflammatory mediators following activation with either IgE/anti-IgE immune complexes or a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Recently, interleukin-10 (IL-10) has been shown to down-regulate various inflammatory responses and to be secreted by lymphocytes and dendritic cells during skin inflammatory reactions. We show here that IL-10 down-regulates the production of tumor necrosis factor (TNF)-α and IL-6 by activated HK. Also, induction of inducible nitric oxide synthase (iNOS) expression in HK by IgE/anti-IgE or LPS/IFN-γ is significantly reduced by the addition of IL-10. This effect is dose dependent and correlates with reduction of iNOS mRNA production and enzyme level. Therefore, IL-10 down-regulates NO-mediated HK inflammatory responses and may thus participate in the regulation of the skin immune network.     

TLR-mediated distinct IFN-γ/IL-10 pattern induces protective immunity against murine visceral leishmaniasis

Resistance to murine visceral leishmaniasis (VL) correlates with the development of an IFN-γ predominant immune response. Beta1,4-galactose terminal glycans are potent inducers of IFN-γ. Here, we demonstrate the efficacy of a 29 kDa β1,4-galactose terminal glycoprotein (GP29) of Leishmania donovani (LD) in an in vitro macrophage model and an in vivo mouse model of VL. GP29 induced splenic macrophages to release NO and ROS in appreciable amounts that resulted in effective parasite clearance from macrophages. This was associated with the toll-like receptor (TLR)-4 mediated IL-12 induction and inhibition of TLR2-mediated IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals resulted in dominant IL-12-mediated IFN-γ production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Vaccinated mice showed a reversal of T-cell anergy, significantly elevated expression of iNOS and a type-1 IgG subclass response. Moreover, vaccinated mice downregulated arginase1 and IL-10 expression but did not alter IL-4 expression. The IFN-γ/IL-10 ratio regulated the intensity of the protective immune response. Experiments with IFN-γ and IL-10 knockout mice reiterated the role IL-10 and IFN-γ play in disease progression or resolution in the murine model of VL.     

Enhanced prostaglandin E2 production by monocytes in atopic dermatitis (AD) is not accompanied by enhanced production of IL-6, IL-10 or IL-12

AD is associated with a bias of the T helper cells to show increased IL-4 and reduced interferon-gamma (IFN-γ) production. The production of IFN-γ and IL-4 and the development of Th cells into either high IFN-γ or high IL-4 producers is strongly influenced by factors produced by antigen-presenting cells (APC), like IL-12 and prostaglandin E₂ (PGE₂). IL-12 selectively enhances IFN-γ production and favours the development of IFN-γ-producing Th cells, whereas PGE₂ selectively inhibits IFN-γ production by Th cells. The aim of this study was to test whether the increased IL-4/IFN-γ production ratio by Th cells in AD can be explained by an increased PGE₂/IL-12 production ratio by the APC. Monocytes were used as APC source. PGE₂ and IL-12 production by lipopolysaccharide (LPS)-stimulated monocytes from 12 AD patients and 12 non-atopic controls was determined using two complementary experimental systems, whole blood cultures and purified monocytes. In addition, we determined IL-6 production as a measure of monocyte activation, and IL-10 production because IL-12 production by monocytes is highly influenced by endogenously produced IL-10. The monocytes from AD patients showed normal production levels of IL-6 and IL-10, a two-fold, but non-significant decrease in IL-12 production, and a significantly (three-fold) higher PGE₂ production than those from non-atopic controls. Here we show for the first time that enhanced PGE₂ production by monocytes in AD is not accompanied by a general rise in cytokine production. We conclude that AD is indeed associated with an increased PGE₂/IL-12 production ratio by monocytes.     

Endogenous nitric oxide and prostaglandin E2 do not regulate the synthesis of each other in interleukin-1β-stimulated rat articular cartilage

Increased levels of nitric oxide (NO) and prostaglandins (PG) are present in the synovial fluid from patients with rheumatoid arthritis and osteoarthritis. Interleukin-1 (IL-1) has been shown to induce the synthesis of both of these mediators. The present work was designed to study the interactions of NO and PGE₂ synthesis induced by IL-1 in rat articular cartilage. Incubation of intact cartilage with IL-1 resulted in different dose response curves for NO and PGE₂ synthesis. Two inhibitors of nitric oxide synthase N-monomethyl-L-arginine (L-NMMA) and L-N-iminoethyl-ornithine, (L-NIO), abolished the IL-1-induced nitrite production but failed to have any influence on the PGE₂ synthesis. Exogenous NO, produced by two chemically different NO-releasing compounds (SIN-1 and GEA 3175) had no effect on PGE₂ synthesis in articular cartilage. Dexamethasone and ketoprofen inhibited IL-1 induced PGE₂ production, while nitrite synthesis remained unaltered. Acetylsalisylic acid (ASA) reduced PGE₂ synthesis and had a slight inhibitory action also on NO production. In conclusion, our results show, that IL-1 induces the synthesis of both PGE₂ and NO in articular cartilage but these two inflammatory mediators are not mediating the synthesis of one another.     

Differential inhibitory effects of indomethacin,dexamethasone, and interferon-gamma (IFN-γ) on IL-11 production by rheumatoid synovial cells

IL-11, a member of the IL-6 type cytokines, has some biological activity related to the joint destruction in rheumatoid arthritis (RA), such as induction of osteoclast differentiation. However, its expression and regulation in rheumatoid inflamed joints has not been clarified. In the present study we examined the capacity of fresh rheumatoid synovial cells (fresh RSC) to produce IL-11, and the effect of indomethacin, dexamethasone and IFN-γ on IL-11 production. Fresh RSC obtained from eight patients with RA produced large amounts of IL-11, measured by ELISA, and showed strong expression of IL-11 mRNA, determined by Northern blotting. Indomethacin inhibited the production of IL-11 by about 55%. Prostaglandin E₂ (PGE₂) completely prevented the inhibition, suggesting that IL-11 production by fresh RSC was in part mediated by PGE₂. Dexamethasone inhibited the production of IL-11 by more than 80%. Interestingly, the inhibition was not abolished by PGE₂. IFN-γ inhibited the production of IL-11 from IL-1α-stimulated cultured rheumatoid synovial fibroblasts, although IFN-γ did not inhibit the production of IL-11 by fresh RSC. These results suggest that the production of IL-11 by rheumatoid synovia was differentially regulated by PGE₂ and IFN-γ, and that treatment with indomethacin or dexamethasone decreased the level of IL-11 at inflammatory joints in patients with RA.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Role of trypanosomatid's arginase in polyamine biosynthesis and pathogenesis

L-Arginine is one of the precursor amino acids of polyamine biosynthesis in most living organisms including Leishmania parasites. L-Arginine is enzymatically hydrolyzed by arginase producing L-ornithine and urea. In Leishmania spp. and other trypanosomatids a single gene encoding arginase has been described. The product of this gene is compartmentalized in glycosomes and is the main source of L-ornithine for polyamine synthesis in these parasites. L-Ornithine is substrate of ornithine decarboxylase (ODC) - one of the key enzymes of polyamine biosynthesis and a validated target for therapeutic intervention - producing putrescine, which in turn is converted to spermidine by condensing with an aminopropyl group from decarboxylated S-adenosylmethionine. Unlike trypanosomatids, mammalian hosts have two arginases (arginase I and II), which have close structural and kinetic resemblances, but localize in different subcellular organelles, respond to different stimuli and have different immunological reactivity. Arginase I is a cytosolic enzyme, mostly expressed in the liver as a pivotal component of the urea cycle, providing in addition L-ornithine for polyamine synthesis. In contrast, arginase II localizes inside mitochondria and is metabolically involved in L-proline and L-glutamine biosynthesis. More striking is the role played by L-arginine as substrate for nitric oxide synthase (NOS2) in macrophages, the main route of clearance of many infectious agents including Leishmania and Trypanosoma cruzi. In infected macrophages L-arginine is catalysed by NOS2 or arginase, contributing to host defense or parasite killing, respectively. A balance between NOS2 and arginase activities is a crucial factor in the progression of the Leishmania infection inside macrophages. In response to T-helper type 2 (Th2) cytokines, resident macrophages induce arginase I inhibiting NO production from L-arginine, thereby promoting parasite proliferation. Conversely, the response to T-helper type 1 (Th1) cytokines is linked to NOS2 induction and parasite death. Moreover, induction of any of these enzymes is accompanied by suppression of the other. Specifically, arginase reduces NO synthesis by substrate depletion, and N(ω)-hydroxy-L-arginine, one of the intermediates of NOS2 catalysis, competitively inhibits arginase activity. In spite of abundant data concerning arginases in mammals as well their involvement in parasite killing, there are very few papers regarding the actual role of arginase in the parasite itself. This review is an update on the recent progress in research on leishmanial arginase including the role played by this enzyme in the establishment of infection in macrophages and the immune response of the host. A comparative study of arginases from other kinetoplatids is also discussed.     

TH2 activated cells prevent experimental autoimmune uveoretinitis,a TH1-dependent autoimmune disease

Mercuric chloride (HgCl₂) injections protect (Lewis x Brown-Norway) F1 (F1) rats against experimental autoimmune uveoretinitis (EAU) induced by immunization with the retinal S antigen (S-Ag); in contrast HgCl₂-injected F1 rats develop EAU following transfer of lymph node (LN) cells from rats immunized with S-Ag alone. In the present study we demonstrate that the ability of LN cells from rats protected against EAU to transfer the disease into naive F1 rats was considerably reduced. These LN cells neither produced interleukin (IL)-2 nor (interferon (IFN)-γ but exhibited mRNA for IL-4. In contrast, LN cells from diseased rats easily transferred EAU into naive F1 rats, produced significant IL-2 and IFN-γ levels but barely exhibited mRNA for IL-4. Furthermore protected rats predominantly produced IgG2b anti-S-Ag antibodies, while diseased rats produced IgG2b anti-S-Ag antibodies and the increase in expression of MHC class II molecules on B cells was higher in protected rats than in diseased rats. These data suggest that (1) to exert a protective effect, HgCl₂ must act at an early stage of differentiation of precursors of S-Ag specific T cells, and (2) this effect is related to the preferential activation of TH2 cells to the detriment of uveitogenic TH1 cells. Finally, these results indicate that activation of TH2 cells protect from a TH1-dependent autoimmune disease.     

黄芪在树突状细胞水平对哮喘TH 1/TH 2平衡的调节作用

目的 探讨黄芪在树突状细胞(DC)水平对过敏性哮喘TH/TH2平衡的调节作用.方法 用rhGM-CSF和rhIL-4诱导培养外周血来源的DC并予鉴定,ELISA法检测其分泌的细胞因子IL-12、IL-10以及与自身T细胞反应后,RT-PCR检测T-bet和GATA-3 mRNA含量,流式细胞术检测T细胞分泌的胞内细胞因子IL-4和IFN-γ水平.结果 哮喘患儿外周血DC分泌IL-10高于对照组(P＜0.05);黄芪干预后DC分泌IL-10降低,与哮喘组比较差异有统计学意义(P＜0.05).哮喘患儿外周血DC分泌IL-12低于对照组(P＜0.05);黄芪干预后DC分泌IL-12增加,但与哮喘组比较差异无统计学意义.混合培养第7天哮喘组T细胞内IL-4水平显著高于正常对照组(P＜0.01);而IFN-γ水平则显著低于正常对照组(P＜0.05);哮喘组IL-4/IFN-γ比值高于正常对照组(P＜0.01).黄芪干预后T细胞内IL-4水平与哮喘组比较差异无统计学意义,而IFN-γ水平增加,与哮喘组比较差异有统计学意义(P＜0.05),IL-4/IFN-γ比值降低,与哮喘组比较差异有统计学意义(P＜0.01).哮喘组T-bet mRNA的表达强度明显低于正常对照组(P＜0.01);而哮喘组GATA-3 mRNA的表达强度则明显高于正常对照组(P＜0.05);哮喘组GATA-3/T-bet比值高于正常对照组(P＜0.05).黄芪干预后T细胞GATA-3 mRNA的表达强度与哮喘组比较差异无统计学意义,而T-bet mRNA水平增加,与哮喘组比较差异有统计学意义(P＜0.05),GATA-3/T-bet比值降低,与哮喘组比较差异有统计学意义(P＜0.01).结论 哮喘患儿DC功能缺陷,产生IL-12减少、IL-10增加导致TH2优势分化,从而使TH1/TH2平衡向TH2倾斜,合成IFN-γ减少,进而造成气道慢性炎症、气道高反应性而致哮喘发作.黄芪对DC的调节主要通过降低IL-10的分泌水平,从而降低其抑制TH0细胞向TH 1分化的功能,即间接抑制了TH0细胞向TH2的分化.     

Induction of macrophage nitric oxide production by interferon-γ and tumor necrosis factor-α is enhanced by interleukin-10

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-γ (IFN-γ)-stimulated macrophages (MΦ) by preventing the secretion of tumor necrosis factor-α (TNF-α) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-γ together with exogenous TNF-α to induce NO synthesis by bone marrow-derived MΦ. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO production by MΦ stimulated in the presence of optimal concentrations of prostaglandin E₂, a positive modulator of MΦ activation by IFN-γ/TNF-α. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-γ/TNF-α cultures and enhanced NO release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on MΦ function are more complex than previously recognized.     

Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans

Mouse peritoneal and splenic macrophages treated with interferon-γ (IFN-γ) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type-2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)-4 and IL-10 on candidacidal activity and NO production of IFN-γ-activated macrophages. Fungal killing and NO secretion were inhibited, in a dose-dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL-4 and IL-10 might involve modulation of NO synthesis.     

恶性淋巴瘤患者TH 1/TH 2细胞因子表达水平的研究

目的 探讨恶性淋巴瘤患者血清中TH1/TH2细胞因子变化及其临床意义,为肿瘤的免疫治疗提供实验依据.方法 用流式细胞小球微阵列术(cytometric bead array,CBA)检测92例恶性淋巴瘤患者及70例健康人群血清中γ干扰素(IFN-γ)、肿瘤坏死因子-α仪(TNF-α)、白细胞介素(IL-2、IL-4、IL-5、IL-10)表达水平.结果 92例恶性淋巴瘤患者血清中TH1型细胞因子的水平分别为:IFN-γ(34.26±33.4g)pg/ml、TNF-α(8.17±10.09)pg/ml、IL-2(3.74 4±1.72)pg/ml;TH2型细胞因子的水平分别为:IL-10(6.28±8.56)pg/ml、IL-5(3.53±3.20)pg/ml、IL-4(6.22±7.13)pg/ml.除TNF-α表达水平降低外,其余5项均明显高于健康体检组,差异有统计学意义(P＜0.01).TH1细胞因子IL-2与TH2细胞因子IL-4的比值明显下降(0.78±O.44),与健康体检组(1.09±0.45)比较差异有统计学意义(P＜0.01).IL-10与疾病的进展相关,Ⅲ/Ⅳ期恶性淋巴瘤患者的表达水平为(9.58±13.96)pg/ml,Ⅰ/Ⅱ期的表达水平为(4.77±3.50)pg/ml,二者比较差异有统计学意义(P＜0.01).IFN-γ在大于60岁的恶性淋巴瘤患者中表达水平明显降低,与其他年龄段恶性淋巴瘤患者比较差异有统计学意义(P ＜0.05).结论 恶性淋巴瘤患者血清中TH1/TH2细胞因子平衡失调,检测TH1/TH2细胞因子可作为评价淋巴瘤临床进展及预后指标.TH1/TH2平衡向TH2方向漂移,这可能是肿瘤细胞发生免疫逃逸,从而导致肿瘤的发生或者转移的原因之一.     

Anti-MHV3 state induced by IFN gamma in macrophages is not related to arginine metabolism

Summary.  In contrast to BALB/c mouse macrophages, the A/J macrophages after activation by interferon gamma (IFN gamma) develop an anti-MHV3 effect which correlates with the resistance to virus infection. To understand the cellular basis of this antiviral effect, we studied the possible involvement of arginine metabolism through nitric oxide (NO) and arginase induction, since these metabolic pathways have been described as implicated in antiviral activities of macrophages. The studies were performed by activating macrophages with inducers of NO (IFN gamma) and arginase (IL4 IL10). NO synthase (iNOS) and arginase inhibitors (N-methyl-arginine, NMA, and hydroxyarginine, OH-ARG) were used. The results show that in both macrophage populations, no spontaneous synthesis of NO occurred and the MHV3 enhanced the NO release induced by IFN gamma. After activation with IFN gamma, BALB/c macrophages released higher amounts of NO than the A/J macrophages. The inhibition of IFN gamma-induced NO-synthesis with NMA or with arginine free medium did not affect the virus replication. In BALB/c macrophages, IL4 or IL10, induced higher amounts of arginase than in A/J macrophages. In both macrophage populations the MHV3 infection had no influence on the arginase synthesized, and the inhibition of the arginase with OH-ARG had no influence on the virus growth. The level of MHV3 replication or inhibition was also not influenced when we used macrophages from knockout mice for the iNOS gene, and as a consequence were unable of synthesizing NO. These data indicate that NO and arginase do not participate in the anti-MHV3 state induced by IFN gamma in macrophages. Received March 4, 1997 Accepted May 14, 1997     

Induction of intracellular arginase activity does not diminish the capacity of macrophages to produce nitric oxide in vitro.

The hypothesis was tested that induction of arginase expression in macrophages (M phi) diminishes nitric oxide (NO) synthesis due to intracellular competition between arginase and inducible nitric oxide synthase (iNOS) for L-arginine (L-arg). Murine M phi cell lines and bone marrow-derived M phi (BMM) were stimulated to express either iNOS or arginase or to co-express these two enzymes. The response pattern obtained was complex but allowed the following conclusions: (i) iNOS and arginase are differentially regulated. (ii) High intracellular arginase levels do not limit the capacity of M phi to synthesize NO even when the L-arg concentration in the culture medium is lowered to physiological levels. (iii) Arginase levels in BMM pre-exposed to either M phi colony-stimulating factor (M-CSF) or granulocyte-M phi colony-stimulating factor (GM-CSF) differ markedly, but iNOS expression and NO synthesis by the two BMM types is similar. (iv) Regulation of iNOS and arginase differs between primary murine bone marrow M phi and murine M phi cell lines. (v) Arginase activity appears to be inhibited during high-output NO synthesis. Taken together, our results show that NO production by M phi is not compromised by conditions that increase intracellular arginase activity.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

Nitric oxide enhances cyclooxygenase activity in articular cartilage

Nitric oxide (NO) is a small messenger molecule synthesized by a family of enzymes, the nitric oxide synthases. Cyclooxygenases are a group of proinflammatory enzymes that release prostaglandins including prostaglandin E₂ (PGE₂). Both nitric oxide synthase and cyclooxygenase are involved in the inflammatory cascade of arthritis. However, the relationship between these two enzymes and their products has not been explored in articular cartilage. Here we show that in cultured bovine chondrocytes and explants of human osteoarthritic cartilage both nitric oxide synthase and cyclooxygenase activities were induced by the inflammatory mediators, lipopolysaccharide, and interleukin-1 or tumor necrosis factor-. When nitric oxide synthase activity was inhibited, PGE₂, synthesis was inhibited. NO donors also induced PGE₂ synthesis and NO scavengers inhibited cyclooxygenase activity. Taken together, these results support the concept that PGE₂ synthesis is directly related to NO formation and that NO may modulate cyclooxygenase activity in articular cartilage.accepted by W. B. van den BergFinalist in the 1995 Westinghouse Science Talent Search, the 1995 Otto Burgdorf Competition, and the 1995 St. Johns New York Symposium.