Impairment in the expression and activity of Fyn during differentiation of naive CD4+ T cells into the Th2 subset.

We previously showed that the amounts of Fyn protein in Th2 clones were approximately one-third to one-fifth of those in Th1 clones. In this study we examined the role of Fyn in the polarization of naive CD4+ T cells toward the Th2 subset using fyn(-/-) mice. The fyn(-/-) naive CD4+ T cells efficiently produced Th2 cytokines and polarized toward the Th2 subset even in the absence of IL-4 and IL-13. The expression of Fyn in wild-type CD4+ T cells decreased at a transcription level concomitant with polarization toward the Th2 subset. These results suggest that Fyn plays a role in the down-regulation of the differentiation of naive CD4+ T cells into the Th2 subset.

A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection.

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.

IL-12 plays a pathologic role at the inflammatory loci in the development of diabetes in NOD mice.

Accumulating evidence suggests that CD4(+)T helper type 1 (Th1) cells play a major role in the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse model. Interleukin (IL)-12 is a potent immunoregulatory molecule that is a key determinant of T-cell differentiation into Th1 cells, and has been implicated in the development of IDDM. To investigate the role of IL-12 that is locally produced by islet-infiltrating cells in the development of IDDM, we generated transgenic NOD mice in which the IL-12 p40 homodimer, a natural antagonist of IL-12, was produced exclusively in islets without affecting the levels of IL-12 p40 in the systemic circulation. We found that the incidence of diabetes was significantly reduced in these transgenic mice. These results clearly demonstrate that IL-12 locally produced by islet-infiltrating cells plays a critical role in the development of IDDM.

Positive and negative regulation of IL-12 receptor expression of naive CD4(+) T cells by CD28/CD152 co-stimulation.

IL-12 is a critical cytokine for polarizing naive CD4(+) T cells toward Th1 subset. Therefore, it is important to elucidate the mechanism of IL-12R expression of naive CD4(+) T cells. In this report, we present evidence to show that expression of both IL-12Rbeta1 and beta2 mRNA is regulated by signals mediated through CD28 and CD152. Naive CD4(+) T cells stimulated with anti-CD3 alone neither expressed IL-12Rbeta2 mRNA nor bound detectable level of rIL-12, although they expressed a very low level of IL-12Rbeta1 mRNA when stimulated with a high dose of anti-CD3. Expression of IL-12Rbeta1 and beta2 mRNA was induced by the co-ligation of CD3 and CD28, and it was down-regulated by the ligation of CD152. CD28 ligation induced not only IL-12Rbeta1 and beta2 mRNA expression, but also enhanced IFN-gammaR to mediate up-regulation of IL-12R by IFN-gamma.

Impaired delayed-type hypersensitivity response in mutant mice secreting soluble CD4 without expression of membrane-bound CD4.

Delayed-type hypersensitivity (DTH) is an important in vivo manifestation of cell-mediated immunity. We examined the DTH response to methylated bovine serum albumin of a novel mutant strain of mice that have soluble CD4 (sCD4) in their circulation without expression of CD4 on the cell surface. The DTH response of the mutant mice was severely impaired, although the response of CD4 knockout (KO) mice, generated by homologous recombination, was comparable to that of wild-type mice. The response of the mutant mice was restored by the neutralization of sCD4 with anti-CD4, and that of CD4KO mice was markedly reduced by the implantation of a diffusion chamber containing sCD4 cDNA transfectant cells. The restored DTH response of the mutant mice treated with anti-CD4 was abolished by treatment with anti-interferon-gamma (IFN-gamma). IFN-gamma production by CD4 mutant and CD4KO mice was consistent with their DTH response and inversely related to the presence of sCD4 in their circulation, indicating that sCD4 impairs the DTH response by blocking the production of IFN-gamma in our mutant mice. These results raise the possibility that sCD4 could impair cell-mediated immunity. Our mutant mice would provide a useful tool with which to analyse the mechanisms of the DTH reaction.

Molecular mechanism of the impairment in activation signal transduction in CD4(+) T cells from old mice.

It is well known that IL-2 production of CD4(+) T cells from old mice (old T cells) is impaired. In this study, we have examined TCR complex zeta chain expression of old T cells and their TCR downstream signal transduction pathways stimulated with anti-CD3. Activation of protein tyrosine kinases, Fyn and ZAP-70, and turnover of inositol phosphates stimulated with anti-CD3 were severely impaired in old T cells, although levels of these proteins were comparable to those in young T cells. Increase in intracellular Ca2+ concentration in old T cells was also impaired. Old T cells starting the Ca(2+) oscillation by the anti-CD3 stimulation were severely decreased in number and the oscillation waves were broader in shape. T cells with zeta-FcvarepsilonRgamma heterodimer in the TCR-CD3 complex were increased in proportion in old T cells with a concomitant decrease in the T cells with zeta-zeta homodimer. The density of the TCR-CD3 complex on old T cells was confirmed to be comparable to that on young T cells. The impairment in TCR downstream signal transduction pathways and the increase in zeta-FcvarepsilonRgamma heterodimer in the TCR-CD3 complex were confirmed to be the situation in Th1 clones established from old mice. These results indicate that old T cells are impaired in response to TCR stimulation, because T cells with the TCR-CD3 complex containing the zeta-FcvarepsilonRgamma heterodimer are increased in proportion in old T cells.

Role of IL-16 in delayed-type hypersensitivity reaction.

Interleukin (IL)-16 is a chemoattractant cytokine for CD4(+) leukocytes. Because delayed-type hypersensitivity (DTH) reaction is mediated by T helper 1 (Th1) cells and CD4(+) T cells can be chemoattracted by IL-16, we have investigated the involvement of IL-16 in the DTH reaction. Immunohistochemical analysis revealed the IL-16 expression in infiltrating cells and epithelial cells in the DTH footpads. The IL-16 expression was also detected intracellularly in the infiltrating cells. In addition, markedly increased production of IL-16 was detected in the DTH footpad extracts, but not in the control footpad extracts, by an enzyme-linked immunosorbent assay and also by Western blot analysis. The DTH footpad extracts exhibited a strong chemoattractant activity toward splenic T cells, which was significantly inhibited by the inclusion of neutralizing monoclonal antibody (mAb) against IL-16 in the migration assay. Furthermore, treatment of sensitized mice in vivo with the anti-IL-16 neutralizing mAb significantly suppressed the footpad swelling induced by an antigen challenge, together with decreased infiltration of leukocytes including not only CD4(+) T cells but also CD8(+) T cells and macrophages into the DTH footpads. Decreased production of macrophage inflammatory protein 1alpha was also observed in the DTH footpad extracts by the mAb treatment. These results suggest that IL-16 plays an important role in the recruitment of leukocytes-presumably including antigen-specific Th1 cells, which secrete cytokines and chemokines mediating the following hypersensitivity reaction after activation by the interaction with Langerhans cells carrying the antigen-for the elicitation of DTH response. (Blood. 2000;95:2869-2874)

Effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells.

We have investigated the effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells to elucidate the role of carbohydrate in the triggering of T cell function. The inhibitors of glycoprotein processing, especially mannnosidase inhibitors, enhanced the anti CD3-induced production of interleukin-2 (IL-2), which is a cytokine without the linkage sequence of N-linked oligosaccharides. On the other hand, N-methyl-1-deoxynojirimycin (NMdNM, an inhibitor of processing glucosidase 1), 1-deoxynojirimycin (dNM, an inhibitor of processing glucosidase I and II) and bromoconduritol (BCD, an inhibitor of processing glucosidase II) inhibited the secretion of interleukin-4 (IL-4), interferon-gamma (IFN-gamma), or interleukin-5 (IL-5) into culture supernatants of anti CD3-stimulated T cells, which had N-linked oligosaccharides. Mannosidase inhibitors, 1-deoxymannojirimycin (dMAN, an inhibitor of processing mannosidase I) and swainsonine (SWN, an inhibitor of processing mannosidase II) did not inhibit the secretion or production of IL-4, IFN-gamma and IL-5. To confirm the inhibition of N-linked oligosaccharide processing in the cytokines by the above inhibitors, the binding of IFN-gamma to lectins with various sugar-binding specificities was investigated. All inhibitors reduced the binding of IFN-gamma to PHA E4, which had a high affinity to bi- or tri-antennary complex type N-linked oligosaccharides with bisecting N-acetylglucosamine. Similarly, all inhibitors reduced the binding of IFN-gamma to PHA L4, which had high affinity to tri- or tetra-antennary complex type N-linked oligosaccharides with beta1-6-linked branching. SWN and dMAN increased the binding of IFN-gamma to concanavalin A (ConA), which had a high affinity to bi-antennary complex type, hybrid type and high-mannose type N-linked oligosaccharides. These results suggest that the processing inhibitors used here inhibit the N-linked oligosaccharide processing of cytokines, and the inhibition of processing enzyme glucosidases I and II induces a decreased secretion of cytokines with N4-linked oligosaccharides.

Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation.

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.

Interferon gamma priming is not critical for IL-12 production of murine spleen cells.

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.

Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling.

Antigenic stimulation of the B-cell receptor (BCR) is a central event in the immune response. In contrast, antigen bound to IgG negatively regulates signals from the BCR by cross-linking it to the inhibitory receptor FcgammaRIIB. Here we show that upon cross-linking of BCR or BCR with FcgammaRIIB, the rasGAP-associated protein p62(dok) is prominently tyrosine phosphorylated in a Lyn-dependent manner. Inactivation of the dok gene by homologous recombination has shown that upon BCR cross-linking, p62(dok) suppresses MAP kinase and is indispensable for FcgammaRIIB-mediated negative regulation of cell proliferation. We propose that p62(dok), a downstream target of many PTKs, plays a negative role in various signaling situations.

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice.

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.

Effective stimulation for IL-12 p35 mRNA accumulation and bioactive IL-12 production of antigen-presenting cells interacted with Th cells.

Bioactive IL-12 is composed of two subunits, p35 and p40. In the APC-Th cell interaction, p40 mRNA accumulation in APC was shown to be up-regulated by stimulation with CD40 ligand (CD40L) on Th cells. However, the CD40-CD40L interaction scarcely induced p35 mRNA accumulation in APC. In the present experiments, p35 mRNA accumulation was induced in splenic macrophages/dendritic cells by the interaction with paraformaldehyde-fixed Th1 cells in the presence of Ag, and the p35 mRNA accumulation was abrogated by the inclusion of anti-I-A in cultures to block TCR/MHC class II interaction. The accumulation was also induced by the stimulation with agonistic anti-I-A. These results indicate that the interaction of the MHC class II molecule with TCR evokes an activation signal for p35 mRNA accumulation in APC. Furthermore, the production of bioactive IL-12 in macrophages/dendritic cells stimulated with CD40L was enhanced by the inclusion of agonistic anti-I-A. The p35 mRNA accumulation and IL-12 production of macrophages/dendritic cells induced by stimulation with OVA-specific fixed Th1 clone expressing CD40L were also enhanced by adding OVA in cultures. These results indicate that the p35 mRNA accumulation induced by MHC class II stimulation plays a role in bioactive IL-12 production.

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical.

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.

Interleukin-12 expression in B cells by transformation with Epstein-Barr virus.

Although interleukin (IL)-12 was originally purified from an Epstein-Barr (EBV)-transformed B cell line and the high correlation of EBV infection and IL-12 expression has been suggested, no study has reported whether EBV infection is directly linked to IL-12 expression. To address this issue, we have investigated IL-12 expression in B cells during in vitro transformation with EBV. Human peripheral B cells became capable of constitutively producing p40 by in vitro transformation with EBV, coincident with the expression of latent membrane protein 1 (LMP1) of EBV. These B cells expressed p40 and p35 mRNA, and phorbol myristate acetate (PMA) stimulation strongly enhanced p40 and p70 production. Furthermore, transfection with LMP1 expression vector into a human B lymphoma cell line, Daudi, led to p40 production with nuclear factor (NF)-kappaB activation. These results suggest that transformation of primary B cells with EBV induces IL-12 expression potentially through LMP1 expression.

Eosinophilia, IgE production, and cytokine production by lung T cells in surface CD4-deficient mutant mice infected with Toxocara canis.

Mutant mice deficient in CD4+ T cells and their normal and heterozygous littermates were infected with Toxocara canis, and compared for eosinophilia, total and Toxocara-specific immunoglobulin E (IgE) production, and in vitro cytokine production by lung cells. The numbers of eosinophils in the peripheral blood of normal and heterozygous mice peaked on days 10 and 21, although mutant mice showed eosinophilia with a peak on day 10. This indicates that the first peak on day 10 is CD4 independent and the second peak is CD4 dependent. Before infection, the levels of total IgE had no significant difference among the three groups of mice. Total and Toxocara-specific IgE in all genotypes of mice increased after infection, and was the highest in normal mice and the lowest in mutant mice. In vitro production of interleukin (IL)-5 and IL-4 by total lung cells was the highest in normal mice and the lowest in mutant mice. CD4+ and CD4- CD8- T lymphocytes, but not CD8+ T lymphocytes produced IL-5 and IL-4 when incubated with anti-CD3 monoclonal antibody (mAb) and lung-adherent cells. These results indicated that IL-5 and IL-4 were produced mainly by CD4+ cells and partly by CD4- CD8- cells, but not by CD8+ cells. In addition, cytokine production by CD4+ cells was affected by the number of CD4 molecules on their surface.

Interleukin-12-dependent mechanisms in the clearance of blood-stage murine malaria parasite Plasmodium berghei XAT, an attenuated variant of P. berghei NK65.

The mechanism of development of host resistance to blood-stage malarial infection was studied by use of an irradiation-induced attenuated variant, Plasmodium berghei XAT, obtained from a lethal strain, P. berghei NK65. The infection enhanced mRNA expression of interleukin (IL)-12 p40 and also of interferon (IFN)-gamma, IL-4, IL-10, and cytokine-inducible nitric oxide synthase (iNOS) in spleen. Treatment of these mice with anti-IL-12 or anti-IFN-gamma led to the progression of parasitemia and fatal outcome. Anti-IL-12 treatment significantly reduced the secretion and mRNA expression of IFN-gamma and greatly diminished the augmentation of iNOS mRNA expression. In addition, recombinant IL-12 administration delayed the onset of parasitemia because of the enhanced IFN-gamma production. These results suggest that blood-stage P. berghei XAT infection induces IL-12 production, which is important for the development of host resistance via IFN-gamma production.

A novel mutant gene involved in T-lymphocyte-specific homing into peripheral lymphoid organs on mouse chromosome 4.

Previously, we have shown a mutant mouse DDD/1 with T-cell-specific homing defect that is regulated by an autosomal recessive gene, plt (paucity of lymph node T cells), and seems to be caused by lymph node (LN) stromal cells. In the present study, immunohistochemical analysis showed unusual distribution of T cells in LN, Peyer's patches (PP), and spleen from plt/plt, probably due to the failure of T cells to migrate from blood into the T-cell zone in LN or PP, or into the spleen white pulp across high endothelial venule or marginal zone, respectively, based on the experiments in which labelled T cells were injected intravenously and detected in the tissues. Analysis of surface L-selectin and CD44 suggested that T cells with memory phenotype, probably from afferent lymphatics, recruit into plt/plt LN. Linkage mapping by simple-sequence length polymorphism of genomic DNA from 190 backcross progenies produced by intercrossing with MSM/Ms, linked plt most closely with D4Mit237, and localized at 24.7 cM from cetromere on chromosome 4. We discuss the possibility that a wild-type gene on plt locus encodes a chemokine inducing T-cell-specific homing into peripheral lymphoid tissues.

A pathogenic role of IL-12 in blood-stage murine malaria lethal strain Plasmodium berghei NK65 infection.

We studied whether the infection with a blood-stage murine malaria lethal Plasmodium berghei NK65 induces IL-12 production, and if so, how the IL-12 production is involved in the protection or pathogenesis. The infection of C57BL/6 mice enhanced mRNA expression of IL-12 p40 and also IFN-gamma, IL-4, and IL-10 in both spleen and liver during the early course of the infection. It also enhanced the mRNA expression of TNF-alpha, Fas ligand, and cytokine-inducible nitric oxide synthase. Increased IL-12 p40 production was also observed in the culture supernatant of spleen cells and in sera of infected mice. In addition, the infection caused massive liver injury with elevated serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and body weight loss. Treatment of these infected mice with neutralizing mAb against IL-12 prolonged the survival and diminished the liver injury with reduced elevation of serum serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities and decreased body weight loss. However, the anti-IL-12 treatment did not affect parasitemia, and all these mice eventually died. Similar results were obtained when infected mice were treated with neutralizing mAb against IFN-gamma. Moreover, anti-IL-12 treatment greatly reduced the secretion and mRNA expression of IFN-gamma in both spleen and liver. These results suggest that the lethal P. berghei NK65 infection induces IL-12 production and that the IL-12 is involved in the pathogenesis of liver injury via IFN-gamma production rather than the protection.