Cognate Th2–B Cell Interaction is Essential for the Autoantibody Production in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a Th2-dominant autoimmune skin disease. We showed that indeed active PV patients had a biased Th2 response and specific IgG4 autoantibodies were dominant. To further investigate the role of antigen-specific Th2 cells in the regulation of pathogenic Dsg3-IgG antibody production, we used recombined Dsg3 protein to immunize wild-type C57BL/6 mice with aluminum hydroxide or complete Freund’s adjuvant as adjuvant. CD4⁺ T cells from Dsg3-immunized mice were adoptively transferred into TCR-β chain deficient mice. The transferred CD4⁺ T cells were readily seen in the peripheral blood and spleen, and interacted with B cells, resulting in B-cell activation. Furthermore, transferred CD4⁺ T cells from mice immunized with Dsg3 plus Alum with Th2 phenotype were able to render unprimed B cells to secrete Dsg3-specific IgG1 antibody in vivo. Taken together, these results provide the first demonstration of direct role of Dsg3-reactive CD4⁺ T (Th2) cells in the regulation of pathologic anti-Dsg3 antibody production.     

Antibody responses to a cytochrome c peptide do not correlate with lymphokine production patterns from helper T-cell subsets.

This paper examines helper T-cell responses and antibody titres and isotypes following immunization with a peptide antigen in association with three different adjuvants. B10.A mice were primed with pigeon cytochrome c fragment 81-104 in association with the adjuvants complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum. Strong antibody responses, dominated by IgG1, were observed upon priming with CFA and IFA. In contrast, priming with alum induced a weak antibody response with little or no detectable antigen-specific IgG1. These differences did not correlate with differences in T-cell priming, as immunization with peptide in association with all three adjuvants induced comparable T-cell proliferative responses and frequencies of antigen-specific cells. In addition, no significant differences in interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-4 production could be found, suggesting that the adjuvants did not differentially affect Th1 and Th2 cells.     

Effect of rolipram, a phosphodiesterase IV inhibitor, on allergic footpad swelling using various adjuvants in mice

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.     

Elicitation of specific, Th1-biased immune response precludes skeletal muscle damage in cruzipain-vaccinated mice

Cruzipain (Cz), the major cystein proteinase of Trypanosoma cruzi, is able to induce protective immunity against parasite challenge. However, some concern has arisen regarding its potential to elicit pathogenic autoimmune reactivity. To determine whether the adverse myopathic effects of Cz-based immunization could be prevented, we evaluated the co-administration of Cz with different adjuvants. Mice were immunized with Cz adjuvantized by alum (Cz+alum), oligodeoxynucleotides containing CpG motifs (Cz+ODN-CpG) or Freund's preparation (Cz+CFA). Cz triggered a vigorous specific humoral response, irrespective of the adjuvant used. Alum mainly drove response towards Th2 phenotype, characterized by specific IgG1 antibodies and IL-10 induction, whereas Cz+ODN-CpG mice exhibited Th1-dominant immunity, with antibodies of the IgG2a isotype and enhanced IFN-gamma production. Histological examination of cardiac tissue demonstrated lesions in Cz+CFA but not in Cz+alum nor Cz+ODN-CpG immunized animals, suggesting that CFA is critical for Cz-mediated injury. Analysis of skeletal muscle revealed that mice receiving Cz+CFA exhibited disrupted and hyalinized myofibers, whereas [Cz+alum]-immunized animals showed hyalinization, architecture modifications and small inflammatory foci. Conversely, no abnormalities were observed in the striated muscle from the Cz+ODN-CpG group. Hence, generation of specific immune response skewed towards Th1, as that recorded for the ODN-CpG adjuvant, may preclude triggering of Cz-mediated muscle tissue damage.     

Interleukin-12 profoundly up-regulates the synthesis of antigen-specific complement-fixing IgG2a,IgG2b and IgG3 antibody subclasses in vivo

The influence of the cytokine interleukin-12 (IL-12) on humoral immune responses was studied in vivo. CBA/J mice immunized with protein antigens (keyhole limpet hemocyanin, phospholipase A₂) adsorbed to aluminum hydroxide (Alum) develop a Th2-like immune response characterized by the production of large amounts of IgG1 as well as some IgE but little IgG2a, IgG2b and IgG3 antibodies. IL-12 is a cytokine that promotes the development and the activation of Th1 cells. Th1 cells are involved in the induction of cellular immunity, which is characterized by low or absent antibody production. On the other hand, some Th1-like immune responses are associated with a strong antibody production of the IgG2a, IgG2b and IgG3 subclasses. Thus, we investigated whether treatment with IL-12 would down-regulate the humoral immune response or stimulate antibody production of the IgG2a, IgG2b and IgG3 subclasses. We observed that: 1) administration of IL-12 to mice together with protein antigens adsorbed to Alum strongly enhanced the humoral immune response by increasing the synthesis of antigen-specific antibodies of the IgG2a, IgG2b and IgG3 subclasses 10- to 1000-fold. The synthesis of IgG1 was not or only slightly (2–5-fold) enhanced, whereas that of the IgE isotype was suppressed. 2) These effects of IL-12 were observed when high (10 μg, 100 μg) or low doses (0.1 μg) of antigen were used for immunization. 3) Titration of IL-12 in vitro revealed that IgG2a is strongly up-regulated over a wide dose range of IL-12 (10 to 1000 ng/day). 4) The effects of IL-12 in vivo are at least partially interferon (IFN)-γ-dependent because an anti-IFN-γ mAb in combination with IL-12 prevented most of the enhanced IgG2a production. 5) Mice receiving IL-12 showed a strong up-regulation of IFN-γ but no inhibition of IL-5 synthesis by spleen cells activated ex vivo with antigen. These results suggest that IL-12 is a potent adjuvant for enhancing humoral immunity to protein antigens adsorbed to Alum, primarily by inducing the synthesis of the complement-fixing IgG subclasses 2a, 2b and 3.     

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30 μg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (> 80% incidence). Although mice immunized with 10 μg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30 μg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.     

Inhibitory peptide analogs derived from a major uveitogenic epitope protect from antiretinal autoimmunity by inducing type 2 and regulatory T cells

We identified inhibitory peptide analogs (IPAs), capable of immunomodulating experimental autoimmune uveitis (EAU), induced in B10.RIII mice by immunization with the retinal antigen interphotoreceptor-binding protein in CFA. Alanine-substituted peptides of the major pathogenic epitope, residues 161-180, were synthesized. They were tested for immunogenicity, cross-reactivity with the native 161-180 epitope, pathogenicity, and ability to prevent EAU when given in IFA before EAU challenge with native murine (m)161-180. Two peptides, 169A and 171A, were unable to elicit disease but cross-reacted with m161-180 by lymphocyte proliferation. Mice pretreated with either of the substituted peptides failed to develop EAU after challenge with the native epitope, m161-180, and had reduced cellular responses by lymphocyte proliferation and by delayed hypersensitivity. Their cytokine response profile to m161-180 showed reduced antigen-specific IFN-gamma and IL-17, whereas IL-4, IL-5, IL-10, and IL-13 from IPA-protected mice were increased, and serum antibody titers to m161-180 revealed reduced IgG2a and elevated IgG1 isotypes, suggesting a Th2 shift in the response. Protection was transferable with lymphoid cells from protected donors to na?ve recipients, who were subsequently immunized for EAU. Thus, IPA pretreatment prevents induction of EAU by skewing the response to a subsequent uveitogenic challenge with the native peptide to a nonpathogenic phenotype, as well as by eliciting transferable regulatory cells.     

Protective immunity against Plasmodium yoelii malaria induced by immunization with particulate blood-stage antigens.

The Plasmodium yoelii murine model was used to test several combinations of blood-stage antigens and adjuvants for the ability to induce immunity to blood-stage malaria. Upon fractionation of whole blood-stage antigen into soluble and insoluble components, only the particulate antigens (pAg) induced protective immune responses. Of a number of adjuvants tested, Quil A was the most effective. Immunization with pAg plus Quil A induced solid protection against nonlethal and lethal P. yoelii challenge infection. Analysis of cytokine production revealed mRNA for Th1-type cytokines (interleukin 2 [IL-2] and gamma interferon) as well as Th2-type cytokines (IL-4 and IL-10) in the spleens of both protected and susceptible animals. The data suggested that the protective pAg response was associated with the earlier production of cytokine mRNA with a Th2 phenotype somewhat favored. Immunization of B-cell-deficient JHD mice indicated that the protection against P. yoelii induced by pAg immunization was B cell dependent. Although immunization with pAg plus Quil A increased the levels of antigen-specific antibodies of all four immunoglobulin G (IgG) isotypes, protection correlated most closely with the presence of IgG1 and IgG2b antibodies. Sera from pAg-plus-Quil A-immunized animals recognized only a limited subset of six to eight distinct P. yoelii antigens, primarily associated with the pAg fraction. These results provide the basis for the identification and characterization of potential vaccine antigens, selected solely for their ability to immunize against blood-stage malaria.     

弗氏佐剂与氢氧化铝佐剂对诱导小鼠获得性免疫应答作用的比较

为研究、比较不同佐剂对诱导小鼠产生获得性免疫应答的不同作用,以卵清白蛋白(OVA)为抗原,分别混合完全弗氏佐剂(CFA)或Al(OH)3佐剂,对C57BL/6小鼠进行常规免疫,采用流式细胞技术对细胞内细胞因子IFN-γ和IL-4进行检测;ELISA方法对特异性抗OVA抗体滴度及抗体亚型进行了检测。结果显示在免疫后CFA组产生以IFN-γ为主的细胞因子而Al(OH)3组产生以IL-4为主的细胞因子;两组中均产生特异性抗OVA IgG抗体,但CFA组以IgG2a亚型为主,而Al(OH)3组则以IgG1亚型为主,不产生IgG2a亚型抗体。实验表明,经CFA加抗原免疫后机体产生的免疫应答以Th1型细胞免疫为主,抗体类型为IgG2a;而Al(OH)3佐剂则诱导机体产生Th2型细胞免疫应答,抗体类型为IgG1。     

Enhanced Th2 immunity after DNA prime-protein boost immunization with amyloid beta (1-42) plus CpG oligodeoxynucleotides in aged rats

Generation and accumulation of fibrillar amyloid beta (Abeta) is widely considered as the pathogenic basis of neurodegeneration in Alzheimer's disease (AD). Both active immunization with fibrillar Abeta and passive immunization with anti-Abeta antibodies in transgenic mouse models of AD result in prevention/dissociation of Abeta plaque formation and restoration of cognitive functions. However, similar immunization studies in humans had to be halted because 6% of the AD patients developed acute meningoencephalitis, likely due to anti-Abeta specific autoimmune Th1 cells. Hence, making Abeta immunotherapy successful requires production of strong antibody responses without Th1-type immunity. In an attempt to develop safer vaccines, we examined the influence of oligodeoxynucleotides as adjuvant on the Th1 and Th2 immune response to Abeta in aged rats. We further investigated whether a DNA prime-protein boost strategy could elicit a more robust Th2 response. The results of the present study showed that all the animals injected with either Abeta peptide alone or Abeta encoding plasmid alone or plasmid DNA prime followed by peptide boost have elicited specific anti-Abeta antibodies. When co-administered, synthetic oligodeoxynucleotides (ODN) further enhanced the anti-Abeta titres. More importantly, the IgG subclasses of the antibodies generated by DNA prime-peptide boost regimen with ODN as adjuvant were primarily of IgG2b and IgG1 isotypes, suggesting that heterologous immunization strategy along with ODN would be advantageous in eliciting more beneficial Th2-type humoral immune response.     

Immune effector cells induced by complete Freund's adjuvant exert an inhibitory effect on antigen-specific type 2 T helper responses

Background II has hecn well documented that environmental factors such as antigenpresenting cells and related cytokines could affect the development of T helper cells. Objective The purpose of this study is to investigate the effect of different adjuvants on T cell development. Methods Ovalbumin (OVA) combined with aluminum hydroxide (Alum) plus pertussis toxin (PT) or complete Freund's adjuvant (CFA) were used to sensitize mice; the production of IgG and IgE anli-OVA antibodies was then followed. In addition, OVA-specific proliferative responses and cytokine production by spleen cells were also investigated. Results The data showed that the adjuvants themselves could modify the pattern of immune response: (1) IgG_2a > anti-OVA antibody was higher in mice sensitized with OVA + CFA compared to that of mice sensitized with OVA + Alum + PT; (2) the ratio of IFN-γ/IL-4 produced by OVA-stimutated spleen cells was higher in mice sensitized with OVA + CFA than that of mice sensitized with OVA + Alum + PT; (3) increased percentage of γδ T cells was noted in the peritoneal exudate cells of OVA + CFA immunized mice; and (4) the immune response of mice sensitized wilh OVA + Alum+ PT was inhibited by the adoptively transferred ascitic cells from OVA + CFA immunized mice. Conclusion In general, the data suggested higher IgG2a and the ratio of IFN-γ/IL-4 was noted In mice sensitized with OVA + CFA. Further elucidation of the regulatory mechanism of allergen-specific T helper cells development and exploration of possible agents for inmiunotherapy might shed light on the management of atopic diseases.     

Pathogenic human monoclonal antibody against desmoglein 3

Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3 (Dsg3), a 130-kDa epidermal cadherin protein. The binding of pathogenic antibody to Dsg3 on epidermal keratinocytes leads to loss of intercellular adhesion and results in intraepithelial blister formation. Here, we describe a human monoclonal antibody, PVMAB786, a Dsg3-specific IgG4 antibody, from an untreated patient with active PV. The antibody reacts with a 130-kDa protein on keratinocyte cell surfaces and recombinant Dsg3 protein, but not desmoglein 1 protein. PVMAB786 induces acantholysis in normal human skin and mucous membranes and induces a clinical and histological profile similar to human PV when injected into neonatal mice. PVMAB786 will be a valuable tool in identifying the role of Dsg3 in epithelial cell adherence and acantholysis, mechanisms of Dsg3 processing/presentation and V gene and isotype usage in PV pathogenesis.     

Epicutaneous immunization converts subsequent and established antigen-specific T helper type 1 (Th1) to Th2-type responses

Epicutaneous immunization is a potential novel technique for topical vaccine delivery. It targets the immunologically rich milieu of the skin while having the advantage of being a non-invasive immunization procedure. By disrupting the stratum corneum of the epidermis a natural adjuvant effect can be achieved through activation of resident Langerhans cells. This negates the normal need for co-application of noxious adjuvants. Epicutaneous immunization on barrier-disrupted skin induces potent antigen-specific systemic immunity with a strong T helper type 2 (Th2) bias. We show here that epicutaneous immunization enhances the vigour of a subsequent T-cell response to the same antigen. The induced systemic Th2 response prevents the development of Th1 responses induced through injection of antigen in complete Freund's adjuvant (CFA). Prior epicutaneous immunization results in reduced production of antigen-specific interferon-gamma and immunoglobulin G2a (IgG2a) and enhanced interleukin-4, IgG1 and IgE responses to immunization with CFA. Moreover, epicutaneous immunization converts an established Th1 response to a Th2 response, as demonstrated by the specific reduction of interferon-gamma and IgG2a and the enhancement of interleukin-4 and IgE. This Th2 dominance of epicutaneous immunization may have direct therapeutic application as an immune-modulating procedure in Th1-dominant diseases such as autoimmune rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis and multiple sclerosis.     

Neither interleukin-6 nor signalling via tumour necrosis factor receptor-1 contribute to the adjuvant activity of Alum and Freund's adjuvant.

The potential contribution made by the inflammatory cytokines, interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) to the adjuvant activity of aluminium hydroxide gels (Alum) or Freund's complete adjuvant (FCA) was studied by comparing the immunological responses of IL-6- or TNF receptor 1- (p55; TNFR-1) deficient mice with immunocompetent control mice. While both TNFR-1- and IL-6-deficient mice primed with ovalbumin (OVA) prepared in either Alum or FCA produced similar IgG.1 responses in comparison to control mice, the pattern of T-helper type 1- (Th1) dependent IgG2a production was significantly altered. In TNFR-1-deficient mice, IgG2a responses were greater than in control mice when FCA, but not when Alum, was used as an adjuvant. Correspondingly, spleen cells from FCA-inoculated TNFR-1-deficient mice restimulated with antigen in vitro produced higher Th1 cytokine (interferon-gamma; IFN-gamma) levels with no alteration in Th2 cytokine (IL-4; IL-5, IL-6 and IL-10) production in comparison with wild-type mice. Higher levels of IgG2a were also detected in IL-6-deficient mice compared with wild-type mice following inoculation with OVA prepared in either FCA or in Alum. Furthermore, analysis of cytokine production by spleen cells revealed that both Th1 and Th2 cytokine production was higher in IL-6-deficient mice compared with control mice. As the majority of the effects of TNF-alpha are mediated via TNFR-1, we conclude that this cytokine inhibits the adjuvant activities of FCA. Furthermore, the results also imply that immunopotentiating effects of FCA or Alum adjuvant are both inhibited by IL-6.     

Therapeutic immunization with radio-attenuated Leishmania parasites through i.m. route revealed protection against the experimental murine visceral leishmaniasis

After our promising results from prophylactic and therapeutic study (i.p. route) with the radio-attenuated Leishmania donovani parasites against experimental murine visceral leishmaniasis, we prompted to check their therapeutic efficacy through i.m route. BALB/c mice were infected with highly virulent L. donovani parasites. After 75 days, mice were treated with gamma (γ)-irradiated parasites. A second therapeutic immunization was given after 15 days of first immunization. The protection against kala-azar was estimated with the reduction of Leishman-Donovan unit from spleen and liver that scored up to 80% and 93%, respectively, while a twofold increase in nitric oxide (NO) and reactive oxygen species (ROS) productions has been observed in the immunized groups of animals. These groups of mice also showed disease regression by skewing Th2 cytokines (IL-10) towards Th1 cytokine (IFN-γ) bias along with the increased generation of NO and ROS, while the infected control group of mice without such treatment surrendered to the disease. Establishment of Th1 ambience in the treated groups has also been supported from the measured antileishmanial antibody IgG subsets (IgG2a and IgG1) with higher anti-soluble Leishmania antigen-specific IgG2a titer. As seen in our previous studies, doses of attenuation by γ-radiation should be taken into serious consideration. Attenuation of parasites at 50 Gy of absorbed dose of gamma rays has not worked well. Thus, therapeutic use of L. donovani parasites radio-attenuated at particular doses can be exploited as a promising vaccine agent. Absence of any adjuvant may increase its acceptability as vaccine candidate further.     

T-cellular autoimmunity against desmogleins in pemphigus,an autoantibody-mediated bullous disorder of the skin

Pemphigus encompasses a group of life-threatening blistering diseases of the skin in which loss of adhesion between keratinocytes is caused by autoantibodies (Ab) against desmogleins (Dsg) 1 and 3. There is major interest in characterizing autoreactive T cells that are presumably critical for the induction and regulation of Ab production. In a recent study, peripheral Dsg3-reactive T helper (Th) cells from patients with acute onset, chronic active and remittent pemphigus vulgaris (PV) were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all the studied PV patients while the number of autoreactive Th1 cells exceeded those of the Th2 cells in chronic active PV. Noteworthy, healthy carriers of the PV-associated HLA class II alleles, DRbeta1*0402 and DQbeta1*0503, exhibited exclusively Th1 reactivity against Dsg3. The titers of Dsg3-reactive IgG were directly related to the ratio of autoreactive Th1/Th2 cells. Moreover, T cell recognition of Dsg3 was restricted by these HLA class II alleles. These findings strongly suggest that (1) Dsg3-reactive Th2 cells are restricted to PV, (2) distinct HLA class II alleles are critical for T cell recognition of Dsg3, and (3) Ab production is associated with both, Th1 and Th2 cells.     

Complex pattern of Th1 and Th2 activation with a preferential increase of autoreactive Th1 cells in BALB/c mice with proteoglycan (aggrecan)-induced arthritis

The central role of CD4+ T cells and the balance between T helper (Th) subpopulations in the pathogenesis of autoimmune diseases have been extensively studied. Proteoglycan (aggrecan)-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA), which is characterized by a Th1 dominance at the onset of the disease. In addition to CD4+ T cells, antigen-presenting B cells and autoantibodies seem to play an important role in the development and regulation of PGIA. To identify proteoglycan-specific CD4+ T cell subsets and Th1- and Th2-supported antibody isotypes during the progression of PGIA, spleen cells of proteoglycan-immunized BALB/c mice were harvested at different times of immunization, and at different stages of the disease, and their cytokine production and antigen-specific antibody isotype profiles were determined by enzyme-linked immunospot (ELISPOT) assays. Both Th1 and Th2 cytokine-producing cells, with the predominance of IL-4/IL-5-secreting cells, were detected during the prearthritic stage, and a shift toward a Th1 dominance was observed at the time of onset of arthritis. Tissue homogenates of acutely inflamed joints contained significantly higher levels of interferon-gamma than IL-4. The prearthritic period and both the acute and chronic phases of joint inflammation were characterized by IgG1 dominance in the sera and this correlated with the number of IgG1-secreting B cells in the spleen. However, the ratio of autoreactive IgG1/IgG2a-secreting cells decreased in arthritic animals. These results indicate the activation and possible regulatory roles of both Th1 and Th2 subsets in the autoimmune process, with the necessity of a relative increase of autoreactive Th1 cells for the induction of joint inflammation.     

Effects of deviating the Th2-response in murine mercury-induced autoimmunity towards a Th1-response

T-helper cells type 1 (Th1) and type 2 (Th2) play an important role in the pathogenesis of autoimmune diseases. In many Th1-dependent autoimmune models, treatment with recombinant interleukin-12 (rIL-12) accelerates the autoimmune response. Mercury-induced autoimmunity (HgIA) in mice is an H-2 regulated condition with antinucleolar antibodies targeting fibrillarin (ANoA), systemic immune-complex (IC) deposits and transient polyclonal B-cell activation (PBA). HgIA has many characteristics of a Th2 type of reaction, including a strong increase of IgE, but disease induction is critically dependent on the Th1 cytokine IFN-gamma. The aim of this study was to investigate if a strong deviation of the immune response in HgIA towards Th1 would aggravate HgIA. Injections of both rIL-12 and anti-IL-4 monoclonal antibody (alpha-IL-4) reduced the HgCl2-(Hg-)induced concentration of the Th2-dependent serum IgE and IgG1, but increased the Th1-dependent serum IgG2a. The IgG-ANoA developed earlier and attained a higher titre after combined treatment, and the ANoA titre of the IgG1 isotype decreased while the ANoA titre of the Th1-associated IgG2a, IgG2b and IgG3-ANoA isotypes increased. Treatment with rIL-12 alone increased the Hg-induced IgG2a and IgG3 ANoA titres, the PBA, and the IC deposits in renal and splenic vessel walls, while treatment with alpha-IL-4 + Hg inhibited renal but not splenic vessel wall IC deposits. We conclude that manipulating the cytokine status, by altering the Th1/Th2 balance, will influence autoimmune disease manifestations. This might be an important way of modulating human autoimmune diseases.     

Profiles of Th1 and Th2 Cytokines after Primary and Secondary Infection by Schistosoma mansoni in the Semipermissive Rat Host

In contrast to most mouse strains, rats eliminate the primary schistosome burden around 4 weeks postinfection and subsequently develop protective immunity to reinfection. In rat schistosomiasis, we have shown predominant expression of a Th2-type cytokine response at the mRNA level after primary infection. In the present study, we showed a significant increase in interleukin-4 (IL-4) mRNA expression in inguinal lymph nodes early after a secondary infection. IL-5 mRNA expression showed a significant increase at days 2 and 4 postreinfection in the spleen and lymph nodes, respectively. We did not detect any gamma interferon (IFN-gamma) mRNA after a challenge infection. Analysis of cytokine secretion by stimulated spleen cells after a primary infection showed predominant expression of IL-4 with maximum production on day 21, accompanied by production of IL-5 from day 11 to day 67. A significant increase in IFN-gamma secretion was detected at day 21. Analysis of immunoglobulin G2b (IgG2b) and IgG2c (Th1-related isotypes) showed undetectable levels of IgG2b, but detectable levels of specific IgG2c antibodies were observed from day 42. The analysis of Th2-related isotypes showed high specific IgG1 and IgG2a antibody titers from day 29. After a secondary infection, only IL-4 and IL-5 secretion was sustained. This is supported by the increased production of Th2-related isotypes. These findings showed that S. mansoni infection can drive Th2 responses in rats in the absence of egg production which is required to induce a Th2 response in mice and are in favor of the role of Th2-type cytokines in protective immunity against reinfection.     

Regulation of Isotype Immunoglobulin Production by Adjuvants in Vivo

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulphate (DXS) in the absence or presence of different adjuvants. The immune response was assayed as the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC) found in various lymphoid organs. The adjuvants influenced the isotype of antibodies produced to the same antigenic determinant. The PFC of different IgG subclasses were favoured by different adjuvants. The IgG3 isotype was produced mainly after immunization with either antigen and lipopolysaccharide (LPS) or Li salt as adjuvant; IgG1 was produced with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), alum, poly I:C, Quil A, Be salt, and poly A:U. Some of the above adjuvants (Be salt and poly A:U) favoured the production of IgG2b, and others (CFA, alum, Quil A, and poly I:C) favoured the IgG2a isotype besides the main isotype. Attempts were made to correlate the activation by the various adjuvants of certain TH subtypes with the isotypes produced.