Differential contributions of B7-1 and B7-2 to the development of murine experimental allergic conjunctivitis

B7-1 and B7-2 are the co-stimulatory molecules that are involved in activation of T cells. We investigated whether B7-1 and B7-2 play a role in the development of T cell-mediated experimental allergic conjunctivitis (EC). EC was induced in Balb/c mice by active immunization with ragweed (RW) followed by RW challenge in eye drops. These mice were treated with neutralizing anti-B7-1 Ab, anti-B7-2 Ab, both Abs, anti-cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) Ab or normal IgGs as controls either during the induction phase or the effector phase. With regard to the induction phase treatment, EC was significantly attenuated when both anti-B7-1 and anti-B7-2 Abs were injected. In contrast, anti-CTLA-4 Ab treatment significantly exacerbated EC. With regard to the effector phase treatment, anti-B7-2 Ab alone significantly attenuated EC, while anti-CTLA-4 Ab tended to exacerbate EC. Collectively, B7-1 and B7-2 differently contribute to the development of EC during the induction and effector phases.     

B7-H3 regulates the development of experimental allergic conjunctivitis in mice

B7-H3 negatively regulates Th1-mediated immune responses. Here, we aimed to investigate whether B7-H3 is involved in the development of murine experimental allergic conjunctivitis (EC), which is predominantly mediated by Th2 cells. Intraperitoneal injection of anti-B7-H3 Ab during the induction phase of EC significantly augmented the severity of EC evaluated as conjunctival eosinophil numbers and Ag-induced IL-5 production by splenocytes. Injection of anti-B7-H3 Ab during the effector phase of EC did not significantly affect the severity of EC. In addition, transfer of Ag-primed splenocytes treated with anti-B7-H3 Ab in vitro did not significantly affect the severity of EC, compared to the splenocytes treated with the control Ab. Thus, regulation of EC by blocking of B7-H3 was observed during the induction phase but not the effector phase. Moreover, this study provides a new notion that B7-H3 regulates not only Th1-mediated but also Th2-mediated immune reactions.     

Endogenous interleukin-10 produced by antigen-irrelevant cells promotes the development of experimental murine allergic conjunctivitis

Interleukin (IL)-10 is known to act as an immunoregulatory cytokine in both T helper cell 1 (Th1)- and Th2-mediated immune responses. Here, we ask whether IL-10 regulates the development of experimental allergic conjunctivitis (EC), a Th2-mediated inflammatory disease. Wild-type (WT) and IL-10 knockout (IL-10 KO) mice were immunized with ragweed (RW) and then repeatedly challenged with RW in eye drops. Twenty-four hours after the final challenge, conjunctivas were harvested for histological analysis, while the blood and spleens were used to determine the RW-specific immunoglobulin levels in serum and proliferation or cytokine responses and splenocyte transfer, respectively. The IL-10 KO mice had significantly less severe EC (as determined by conjunctival eosinophil infiltration) than the WT mice and evinced greater RW-specific splenocyte proliferation and cytokine production. However, the RW-specific immunoglobulin levels of the two strains did not differ. When the splenocytes from RW-primed WT mice were transferred into IL-10 KO or WT mice, the IL-10 KO mice showed significantly less conjunctival eosinophil infiltration. In contrast, when the splenocytes from RW-primed IL-10 KO or WT mice were transferred into WT mice, both splenocyte populations generated equivalent severe EC. These data indicate that IL-10 does not serve as an immunoregulatory cytokine in the development of EC. Instead, it appears that IL-10 produced by antigen-irrelevant cells acts in the effector phase to promote the development of EC.     

CD27 and CD70 do not play a critical role in the development of experimental allergic conjunctivitis in mice

CD27, which belongs to the TNF receptor family, is a costimulatory molecule that participates in T-cell activation. Unlike costimulatory molecules such as OX40 and 4-1BB, little is known about the role CD27 plays a role in the development of experimental diseases. We asked whether CD27 and its ligand CD70 participate in the development of experimental allergic conjunctivitis (EC) in BALB/c mice, which is generated by immunization with ragweed (RW) in alum and challenged 10 days later with RW in eye drops. The roles of CD27 and CD70 were tested by intraperitoneally injecting the mice with anti-CD27, anti-CD70 or a control Ab during the induction or effector phase. Twenty-four hours after challenge, the conjunctivas, blood and spleens were harvested for histological analysis, measuring Ig levels and cytokine analysis, respectively. Regardless of when the mice were treated, anti-CD27 or anti-CD70 Ab treatment did not significantly affect the severity of EC as evaluated by conjunctival eosinophil numbers. However, anti-CD27 or anti-CD70 Ab treatment during the induction phase did significantly modulate systemic humoral and cellular immune responses. In vitro treatment of RW-primed splenocytes with anti-CD27 or anti-CD70 Ab did not affect the EC-inducing capability of the splenocytes. Taken together, CD27 and CD70 do not play a critical role in the development of EC.     

Do T helpers 1 and 2 recognize different class II MHC molecules? Humoral and cellular immune responses to soluble allergen from Aspergillus fumigatus Asp f2

Cellular signals leading to T helper (Th)1/Th2 shift are not well known. Here we demonstrate that Th1 possibly recognizes peptides presented by the IE molecule of MHC class II while Th2 is activated by the recognition of peptides presented by the IA molecule. BALB/c mice immunized with Asp f2 developed stable IA-restricted Th2 immune response to the 12th day after immunization, as analyzed by IL-2 production. On the contrary, early Th0 cells did not secrete IL-2 upon Asp f2 stimulation but did produce a high level of IL-2 if stimulated in the presence of anti-IA Abs. This effect of anti-IA Abs on early Th0 cells was both MHC IE and CD4(+) cell restricted. In vivo blocking of Asp f2 peptide presentation by the IA molecule led to the formation of antigen-specific cytotoxicity as demonstrated using immune splenocytes as effector cells and Asp f2 loaded P815 cells as targets.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH ALCOHOL

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice splenocytes and thymocytes depended on the presence of the oncogene product, on the in vivo pretreatment with alcohol, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes released less sIL-2R than FVB thymocytes. Alcohol did not increase sIL-2R release in Oncomice as it did in FVB mice thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes, their secretion was downregulated by in vivo treatment with alcohol, while it was upregulated in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes from animals receiving alcohol. Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. Therefore, the in vivo treatment with alcohol modified the in vitro response to cocaine or morphine in an oncogene-dependent and -independent manner. Hence, our results further emphasize the role of v-Ha-ras oncogene in defining the host immune response, and of alcohol in modulating such response.     

IN VITRO RESPONSE OF v-Ha-ras TRANSGENIC MOUSE LYMPHOCYTES AFTER IN VIVO TREATMENT WITH COCAINE

Oncomouse is a transgenic mouse carrying an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter. The objective of this paper was to learn if the in vitro secretion of IL-2 and IFN-γ and the release of sIL-2R by Oncomice spleen and thymus cells depended on the presence of the oncogene product, on the in vivo pretreatment with cocaine, or on the in vitro treatment with cocaine or morphine. Oncomice thymocytes from different experimental groups released less sIL-2R than FVB thymocytes. Oncomice thymocytes secreted more IFN-γ than FVB thymocytes. Oncomice thymocytes cultured in the presence of Con A and cocaine showed a diminished release of sIL-2R and a lower secretion of IFN-γ, a phenomenon not observed in FVB thymocytes. IFN-γ secretion was lower in Oncomice splenocytes. In general, Oncomice thymocytes and splenocytes responded in a nearly opposite fashion to their FVB counterparts. In this study, the in vitro response to mitogens, cocaine or morphine depended on genetic background and not on the in vivo pretreatment with cocaine. Our results emphasize the role of the v-Ha-ras oncogene in defining the host immune response.     

Prevention of experimental autoimmune uveoretinitis by monoclonal antibody to interleukin-12

Experimental autoimmune uveoretinitis (EAU) induced by immunization with interphotoreceptor retinoid-binding protein (IRBP), a retinal self antigen, has been regarded to be a typical T helper type 1 (Th1)-mediated inflammatory disease. In this study, we examined the effect of a neutralizing monoclonal antibody (mAb) to interleukin-12 (IL-12), which has been known to play a critical role in the Th1 differentiation, on the development of EAU. While 9 of 13 control mice developed EAU by the immunization with IRBP, none of 12 mice developed EAU when given anti-IL-12 mAb 1 day before immunization. These mice did not develop EAU even after a rechallenge with IRBP on day 30, indicating that a protective mechanism had been established by the anti-IL-12 treatment. The proliferative response of splenocytes to IRBP in vitro was not significantly impaired, but the production of IL-2 and IFN-γ was greatly reduced by the anti-IL-12 treatment. Moreover, production of IL-5 and expression of IL-4 mRNA were increased by the anti-IL-12 treatment. Consistently, IgG2a anti-IRBP serum antibodies were decreased and IgG1 were increased. Administration of a neutralizing anti-IL-4 mAb at the time of IRBP rechallenge reversed the protection established by the anti-IL-12 treatment at the primary immunization. These results indicate that the anti-IL-12 treatment at the IRBP priming not only prevented the development of pathogenic Th1 cells, but also induced suppressive Th2 cells that protect the animals from further challenge with the same antigen.     

Modulation of murine experimental allergic conjunctivitis by treatment with alpha-galactosylceramide

When mice are treated with alpha-galactosylceramide (alpha-GalCer), NKT cells are activated and suppress the development of experimental airway inflammation. This suppressive effect is believed to be mediated by the upregulation of IFN-gamma. Here, we investigated whether alpha-GalCer treatment can also modulate the development of experimental allergic conjunctivitis (EC). EC was induced in wild-type and IFN-gamma-deficient Balb/c mice by active immunization with ragweed (RW) followed by challenge with RW in eye drops. The mice were intraperitoneally injected with alpha-GalCer or vehicle at the time of immunization or before RW challenge. Twenty-four hours after RW challenge, conjunctivas, spleens and sera were harvested for histological analysis, flow cytometric, proliferation and cytokine assays, and measurement of immunoglobulin levels, respectively. Treatment with alpha-GalCer at the time of the EC-priming immunization significantly increased Th2 responses and markedly upregulated the severity of the EC. However, treatment with alpha-GalCer just before the Ag challenge that triggers EC in primed animals significantly suppressed the disease. This was associated with an increased frequency of CD4(+)CD25(+) cells, which express Foxp3, in the spleen. alpha-GalCer treatment just prior to Ag challenge also suppressed the development of EC in IFN-gamma-deficient mice, and we found apoptosis and anergy are unlikely to play a major role in the mechanism by which pre-challenge alpha-GalCer treatment suppresses EC. These data suggest that NKT cells can play a downregulatory role in the development of EC and that alpha-GalCer may be useful for treating allergic conjunctivitis.     

Increase of Natural Killer Activity of Mouse Lymphocytes Following in vitro and in Vivo Treatment with Lithium

The in vivo and in vitro influence of lithium lactate on mouse natural killer activity was investigated. In vitro exposure of effector-target mixture to graded concentrations of lithium did not substantially modify the natural killer activity of mouse splenocytes, untreated or pretreated with cyclophosphamide. However in vitro treatment of effector splenocytes increased the frequency of NK-percursor cells.

The in vivo treatment with lithium lactate greatly increased the natural killer activity in intact mice, whereas it did not improve this cytotoxic function in host immunodepressed by cyclophosphamide.

These data suggest that lithium salts produce a modulation of natural killer activity of mouse spleen cells, probably through a mechanism involving the increase of the number of NK-precursors in hosts not subjected to cytotoxic chemotherapy.     

Mycobacterium vaccae immunization to OVA sensitized pregnant BALB/c mice suppressed placental and postnatal IL-5 and inducing IFN-gamma secretion

Although the development of atopy in the newborn is determined by a multitude of factors, an intense Th1 stimulus early in life could be protective by facilitating a switch away from Th2. Aimed to determine the effect of single Mycobacterium vaccae (M. vaccae) immunization to OVA-sensitized pregnant mice on IL-5 and IFN-γ secretion from placental lymphocytes and splenocytes of offspring.

Pregnant BALB/c mice were divided into 4 groups, OVA-sensitized + M. vaccae immunized, OVA-sensitized, M. vaccae immunized and controls. Sensitization with OVA was initiated before mating, and aerosol OVA challenge were performed during pregnancy. M. vaccae immunization was performed on the 12^th day of pregnancy. IL-5 and IFN-γ levels of placental lymphocytes were analyzed on the 18^th day of pregnancy and splenocytes of offspring on the 2^nd and 28^th days during postnatal period. A single administration of M. vaccae to OVA-sensitized pregnant mice downregulated IL-5 secretion and induced IFN-γ secretion from placental lymphocytes. On the other hand, after M. vaccae immunization downregulation of IL-5 levels and upregulation of IFN-γ secretion persisted in offspring when determined on 2^nd and 28^th days of life. Vaccination with M. Vaccae to OVA-sensitized pregnant BALB/c mice prevented Th2 immune responses by enhancing secretion of IFN-γ and lowering IL-5 levels during pregnancy and the effect persisted during the postnatal period in offspring.     

Blockade of CD70-CD27 Interaction Inhibits Induction of Allergic Lung Inflammation in Mice

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T-cell activation. In this study, we investigated the effects of neutralizing anti-CD70 monoclonal antibody (mAb) in a murine model of allergic lung inflammation to determine whether CD27 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. BALB/c mice were immunized by an injection of ovalbumin (OVA) with alum adjuvant and challenged with aerosolized OVA in PBS. Some groups of mice were treated with anti-CD70 mAb or control rat IgG during the induction or effector phase. The administration of anti-CD70 mAb during the induction phase, but not the effector phase, reduced eosinophil infiltration in lung tissue compared with control IgG-treated mice. Treatment with anti-CD70 mAb also resulted in the decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the bronchoalveolar lavage fluid and draining lymph node cell cultures. We further revealed that antigen-specific CD4 T cells were separated into CD27(+) and CD27(-) populations in the lymph nodes of OVA-immunized DO11.10/Rag-2(-/-) mice. The CD27(+) CD4 T cells produced a high concentration of IFN-γ, representing Th1 cells. In contrast, CD27(-) CD4 T cells produced high concentrations of IL-4, IL-5, and IL-13, representing Th2 cells. Moreover, the population of CD27(-) Th2 cells was significantly reduced by the anti-CD70 mAb treatment. These results indicate an important role for CD27 in the development of pathogenic Th2 cells in a murine model of allergic lung inflammation.     

CCK-8对KLH免疫小鼠脾细胞Th1/Th2平衡的影响

目的： 探讨八肽胆囊收缩素（CCK-8）对Th1/Th2平衡的调节作用。方法: 给予BALB/c小鼠钥孔戚血蓝蛋白（KLH）免疫同时体内给予不同剂量的CCK-8,酶联免疫吸附试验(ELISA)检测其脾细胞培养上清中Th1型细胞因子γ-干扰素(IFN-γ)、白细胞介素-2（IL-2）和Th2型细胞因子白细胞介素-4（IL-4）、白细胞介素-5（IL-5）水平,逆转录聚合酶链式反应(RT-PCR)法检测脾细胞中IFN-γ、IL-2、IL-4、IL-5 mRNA表达;ELISA法检测血清中Th1型抗KLH抗体IgG2a和Th2型抗KLH抗体IgG1水平。结果: ①KLH免疫使小鼠脾细胞分泌Th1/Th2型细胞因子水平明显增高,mRNA表达增高,KLH免疫同时给予CCK-8可使脾细胞培养上清中IFN-γ、IL-2含量进一步增加和IFN-γ、IL-2mRNA表达增高,而使IL-4、IL-5含量降低,IL-4、IL-5 mRNA表达减低和降低IL-4/IFN-γ比值。②KLH免疫小鼠血清中IgG2a、IgG1发生不同程度增高,CCK-8可使其血清中IgG1水平减低而使IgG2a水平增高。结论: CCK-8可促进KLH免疫小鼠体内Th1反应,使Th2优势反应向Th1方向转变。     

Role of antigen-presenting cells in the polarized development of helper T cell subsets: evidence for differential cytokine production by Th0 cells in response to antigen presentation by B cells and macrophages

Immune challenges can elicit polarized responses skewed towards the development of T helper type 1 (Th1) or Th2 T cell subsets. To determine if distinct antigen-presenting cells (APC) populations might selectively influence Th subset development, we studied the role of two key APC populations, B cells and macrophages, in the differentiation of effector Th populations from naive precursor Th in vitro. Antigen (Ag)-specific, naive CD4⁺ T cells were enriched from a mouse strain, AND, bearing a transgenic α/β T cell receptor (TCR) encoding reactivity with pigeon cytochrome c peptide 88-104. Peptide Ag was used throughout these studies so that differences in the uptake and processing by the two APC populations would not influence the results. Both APC populations, activated B cells and bone marrow-derived macrophages, supported the development of effector Th having the capacity to secrete high levels of cytokines when restimulated. Regardless of APC population present during effector development, exogenous interferon-γ (IFN-γ) and interleukin-4 (IL-4) had dominant effects on Th subset development. Thus, with both APC populations, effector Th generated in the presence of IFN-γ acquired a Th1-type cytokine profile, Th generated with IL-4 acquired a Th2-type cytokine profile, and Th generated without IFN-γ or IL-4 acquired a Th0-type cytokine profile. B cells and macrophages also had equivalent APC function in the restimulation of Th1 and Th2-like effectors, since only minor differences in cytokine production were noted for these effector populations when restimulated with the two APC populations. However, in 8 of 19 experiments, the Th0-like effector population generated in the presence of IL-2 differentially responded to restimulation with B cells and macrophages, secreting significantly more IFN-γ when restimulated with B cells, and significantly more IL-4 when restimulated with macrophages. We also found that Th effector populations recultured in IFN-γ or IL-4 assumed a more Th1 or Th2-like phenotype, respectively, regardless of their initial cytokine profile. We conclude that through a subtle capacity to skew cytokine production by a Th0 subset, different APC may selectively influence Th subset development under conditions of prolonged or chronic stimulation in an autocrine fashion.     

乳杆菌对原代淋巴细胞中Th1/Th2细胞平衡的影响

目的:分析7种乳杆菌对原代淋巴细胞增殖和细胞因子(CK)分泌的作用,进而探讨其对Th1/Th2细胞平衡的影响。方法:用不同种属、不同浓度的活的/热致死的乳杆菌体外作用于小鼠脾淋巴细胞培养60 h后,采用MTT比色法检测淋巴细胞的增殖效果。用ELISA法检测Th1型细胞因子(IL-12、IFN-γ)、Th2型细胞因子(IL-4、IL-10)和调节型细胞因子(TGF-β)的分泌量。结果:活的/热致死的乳杆菌单独作用,就能促进淋巴细胞体外增殖并表现出剂量依赖关系(P<0.05)。当菌的浓度为107集落形成单位(CFU)/mL(即细菌与细胞的比例为10∶1)时,热致死的发酵乳杆菌和嗜酸乳杆菌的免疫活性近似于活菌。而且,这两株热致死菌还可适当提高淋巴细胞分泌IL-12和IFN-γ,抑制IL-4、IL-10和TGF-β的分泌,使其IFN-γ/IL-4的比值(代表Th1/Th2细胞平衡)均显著高于刀豆蛋白A(ConA)对照组(P<0.05)。结论:乳杆菌可通过提高淋巴细胞的IFN-γ/IL-4分泌率来促进Th1优势状态的Th1/Th2细胞平衡,并具有菌株特异性。     

Chlamydia trachomatis infection inhibits airway eosinophilic inflammation induced by ragweed.

While much progress has been achieved in controlling infectious diseases, there is a startling increase in the prevalence of allergic disorders in developed countries. Previous studies using experimental murine models of asthma have demonstrated that mycobacterial infections are capable of suppressing asthma-like reactions induced by ovalbumin (OVA). Using a different intracellular bacterium, Chlamydia trachomatis mouse pneumonitis (MoPn), we examined the effect of infection on the development of allergic responses to a common natural airborne allergen, ragweed (RW). The data showed that airway eosinophilia induced by ragweed sensitization/challenge was significantly reduced in MoPn-infected mice. MoPn-infected mice also exhibited significantly lower levels of allergen-driven Th2 cytokine production, namely IL-4, IL-5, IL-10, and IL-13, following ragweed exposure in comparison with those treated with ragweed only. Additionally, the production of eotaxin, a C-C chemokine for eosinophil chemoattraction following RW exposure, was significantly reduced in the lungs of MoPn-infected mice. However, MoPn infection did not reduce the levels of RW-specific IgE and IgG1 production in the sera, nor did it diminish the level of total serum IgE. These data provide evidence that the suppression of the allergic airway inflammation induced by a common environmental allergen is attainable through intracellular bacterial infection.     

IL-2 may be a limiting factor precluding lymphocytes from genetically resistant mice from responding to HgCl2.

It is unclear how HgCl2 causes autoimmune disorders in genetically predisposed rodents. We investigated the cytokine profile induced by HgCl2 in vitro, and found a high frequency of IL-2-secreting cells in splenocytes from susceptible A.SW and BALB/c mice, whereas the frequency was low in cells from resistant DBA/2 mice. More IL-2-secreting cells were induced in splenocytes from the high responder A.SW mice than in cells from the intermediate responder BALB/c mice. Unexpectedly, a similar level of IL-4 production was induced in splenocytes from BALB/c and DBA/2 mice. IL-4 production was high in unstimulated cells from A.SW mice and was further increased by HgCl2. IFN-gamma-secreting cells were detectable in splenocytes from all three strains after activation by HgCl2. The highest frequency of IL-10-secreting cells was found in splenocytes from A.SW mice after activation, whereas the frequency was lower in cells from BALB/c mice, followed by cells from DBA/2 mice. We showed that neutralizing anti-IL-2 antibody profoundly inhibited the in vitro response to HgCl2. In contrast, antibodies against IL-4, IFN-gamma and IL-10 did not significantly affect the response of splenocytes from either A.SW or DBA/2 mice. The addition of IL-2 into cultures enhanced the proliferative response to HgCl2 in splenocytes from DBA/2 mice to a level comparable with that in cells from BALB/c mice. We found no evidence for the suggestion that HgCl2 induces a Th1/Th2 imbalance in resistant/susceptible strains. We conclude that IL-2 may be a limiting factor precluding lymphocytes from resistant mice from responding to HgCl2.