Dichotomy of autoreactive Th1 and Th2 cell responses to desmoglein 3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles

Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells.     

Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. Previously, we have generated an active mouse model for PV by adoptive transfer of Dsg3(-/-) splenocytes. In this study, we isolated eight AK series, anti-Dsg3 IgG mAbs from the PV mouse model, and examined their pathogenic activities in induction of blister formation. Intraperitoneal inoculation of the AK23 hybridoma, but not the other AK hybridomas, induced the virtually identical phenotype to that of PV model mice or Dsg3(-/-) mice with typical histology of PV. Epitope mapping with domain-swapped and point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognized a calcium-dependent conformational epitope on Dsg3, which consisted of the V3, K7, P8, and D59 Dsg3-specific residues that formed the adhesive interface between juxtaposed Dsg, as predicted by the crystal structure. The epitopes of the mAbs that failed to show apparent pathogenic activity were mapped in the middle to carboxyl-terminal extracellular region of Dsg3, where no direct intermolecular interaction was predicted. These findings demonstrate the pathogenic heterogeneity among anti-Dsg3 IgG Abs due to their epitopes, and suggest the direct inhibition of adhesive interaction of Dsg as an initial molecular event of blister formation in pemphigus.     

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.     

Dissecting the anti-desmoglein autoreactive B cell repertoire in pemphigus vulgaris patients

Pemphigus vulgaris (PV) encompasses two clinical phenotypes, one producing mucosal blisters and the other mucosal and skin lesions (mcPV). The mucosal blister-producing PV variant is characterized by autoantibodies against desmoglein (Dsg)3, whereas mucosal and skin lesion-producing PV is characterized by autoantibodies to Dsg3 and Dsg1. The present study was aimed at disclosing the diversity and clonality of the anti-Dsg3 response, as well as whether anti-Dsg3 B cells are Ag selected. Human-mouse heterohybridomas were generated by fusion of EBV-transformed or freshly isolated PBLs from six PV patients with mouse myeloma cells. A total of 73 anti-Dsg hybridomas (47 IgM and 26 IgG) were isolated. Over 90% are specific for both Dsg1 and Dsg3 indicating extensive cross-reactivity between these responses. V(H) gene segment use by IgM hybridomas is diverse, but is restricted among IgG hybridomas, where the majority uses one of two V(H) genes. V(L) gene segment use was diverse even among IgG hybridomas suggesting that the V(L) is less critical to defining desmoglein specificity. Additionally, the IgG hybridomas were extensively mutated and the distribution and nature of the mutations suggested that they had been Ag selected. We conclude that the potentially pathogenic IgG anti-Dsg response is restricted in V(H) use, is somatically mutated, and is Ag selected.     

Anti-desmoglein 3 (Dsg3) monoclonal antibodies deplete desmosomes of Dsg3 and differ in their Dsg3-depleting activities related to pathogenicity

Pemphigus vulgaris (PV) is an autoimmune blistering disease, characterized by the loss of cell-cell adhesion between epidermal keratinocytes and the presence of autoantibody against desmoglein 3 (Dsg3), which provides adhesive integrity to desmosomes between adjacent keratinocytes. We have previously shown that PV-IgG purified from patients depletes desmosomes of Dsg3. However, PV-IgG contains not only antibodies against a variety of different epitopes of Dsg3 but also against other unknown antigens. Therefore, we examined whether the Dsg3-depleting activity of PV-IgG is generated specifically by anti-Dsg3 activity in a human squamous cell carcinoma cell line (DJM-1) and normal human keratinocytes by using four different pathogenic and nonpathogenic monoclonal antibodies against Dsg3. We demonstrate that these monoclonal antibodies deplete cells and desmosomes of Dsg3, as PV-IgG does. Individual monoclonal anti-Dsg3 antibodies display characteristic limits to their Dsg3-depleting activity, which correlates with their pathogenic activities. In combination, these antibodies exert a cumulative or synergistic effect, which may explain the potent Dsg3-depleting capability of PV-IgG, which is polyclonal. Finally, although Dsg3-depletion activity correlated with AK-monoclonal antibody pathogenicity in mouse models, the residual level of Dsg3, when below approximately 50%, does not correlate with the adhesive strength index in the present study. This may suggest that although the Dsg3 depletion is not indicative for adhesive strength, the level of Dsg3 can be used as a read-out of pathogenic changes within the cell and that the Dsg3 depletion from desmosomes plays an important role in skin fragility or susceptibility to blister formation in PV patients.     

Signaling Dependent and Independent Mechanisms in Pemphigus Vulgaris Blister Formation

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.     

Type I regulatory T cells specific for desmoglein 3 are more frequently detected in healthy individuals than in patients with pemphigus vulgaris

Pemphigus vulgaris (PV) is a severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies against desmoglein 3 (Dsg3) that are presumably regulated by Th cells. The aim of this study was to identify Dsg3-specific T regulatory (Tr) cells that may help to maintain and restore natural tolerance against Dsg3. Dsg3-responsive IL-10-secreting Tr1 cells were isolated by MACS cytokine secretion assay from healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, but were only rarely detected in PV patients. The Dsg3-specific Tr1 cells secreted IL-10, TGF-beta, and IL-5 upon Ag stimulation, proliferated in response to IL-2 but not to Dsg3 or mitogenic stimuli, and inhibited the proliferative response of Dsg3- and tetanus toxoid-responsive Th clones in an Ag-specific (Dsg3) and cell number-dependent manner. Moreover, their inhibitory effect was blocked by Ab against IL-10, TGF-beta, and by paraformaldehyde fixation. These observations strongly suggest that 1) Dsg3-responsive Tr1 cells predominate in healthy individuals, 2) their growth requires the presence of IL-2, and 3) they exert their Dsg3-dependent inhibitory function by the secretion of IL-10 and TGF-beta. Because autoaggressive T cells responsive to identical epitopes of Dsg3 were recently found both in PV patients and healthy individuals, the identified Tr1 cells may be critically involved in the maintenance and restoration of tolerance against Dsg3.     

Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1-496) and Dsg3 (1-566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26-87 of Dsg1 and 25-88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His(25), Cys(28), Ala(29)) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.     

Cutting Edge: Brazilian Pemphigus Foliaceus Anti-Desmoglein 1 Autoantibodies Cross-React with Sand Fly Salivary LJM11 Antigen

The environmental factors that contribute to the development of autoimmune diseases are largely unknown. Endemic pemphigus foliaceus in humans, known as Fogo Selvagem (FS) in Brazil, is mediated by pathogenic IgG4 autoantibodies against desmoglein 1 (Dsg1). Clusters of FS overlap with those of leishmaniasis, a disease transmitted by sand fly (Lutzomyia longipalpis) bites. In this study, we show that salivary Ags from the sand fly, and specifically the LJM11 salivary protein, are recognized by FS Abs. Anti-Dsg1 monoclonal autoantibodies derived from FS patients also cross-react with LJM11. Mice immunized with LJM11 generate anti-Dsg1 Abs. Thus, insect bites may deliver salivary Ags that initiate a cross-reactive IgG4 Ab response in genetically susceptible individuals and lead to subsequent FS. Our findings establish a clear relationship between an environmental, noninfectious Ag and the development of potentially pathogenic autoantibodies in an autoimmune disease.     

Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a life-long, potentially fatal IgG autoantibody-mediated blistering disease targeting mucocutaneous keratinocytes (KCs). PV patients develop pathogenic anti-desmoglein (Dsg) 3 ± 1 and antimitochondrial antibodies (AMA), but it remained unknown whether and how AMA enter KCs and why other cell types are not affected in PV. Therefore, we sought to elucidate mechanisms of cell entry, trafficking, and pathogenic action of AMA in PV. We found that PVIgGs associated with neonatal Fc receptor (FcRn) on the cell membrane, and the PVIgG-FcRn complexes entered KCs and reached mitochondria where they dissociated. The liberated AMA altered mitochondrial membrane potential, respiration, and ATP production and induced cytochrome c release, although the lack or inactivation of FcRn abolished the ability of PVIgG to reach and damage mitochondria and to cause detachment of KCs. The assays of mitochondrial functions and keratinocyte adhesion demonstrated that although the pathobiological effects of AMA on KCs are reversible, they become irreversible, leading to epidermal blistering (acantholysis), when AMA synergize with anti-Dsg antibodies. Thus, it appears that AMA enter a keratinocyte in a complex with FcRn, become liberated from the endosome in the cytosol, and are trafficked to the mitochondria, wherein they trigger pro-apoptotic events leading to shrinkage of basal KCs uniquely expressing FcRn in epidermis. During recovery, KCs extend their cytoplasmic aprons toward neighboring cells, but anti-Dsg antibodies prevent assembly of nascent desmosomes due to steric hindrance, thus rendering acantholysis irreversible. In conclusion, FcRn is a common acceptor protein for internalization of AMA and, perhaps, for PV autoantibodies to other intracellular antigens, and PV is a novel disease paradigm for investigating and elucidating the role of FcRn in this autoimmune disease and possibly other autoimmune diseases.     

Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG.Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin- and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin- and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.     

IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice

We have previously shown that naive SJL (H-2(s)) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP(139-151)-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP(139-151)-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP(139-151)-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP(139-151)-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10(-/-) mice with high numbers of PLP(139-151)-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP(139-151) peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.     

STAT3 and NF-kappaB signal pathway is required for IL-23-mediated IL-17 production in spontaneous arthritis animal model IL-1 receptor antagonist-deficient mice

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.     

Changes in desmoglein 1 expression and subcellular localization in cultured keratinocytes subjected to anti-desmoglein 1 pemphigus autoimmunity

The complexity of pemphigus acantholysis together with the weak expression of desmoglein 1 (Dsg1) in cultured keratinocytes have made the study on the pathogenic action of anti-Dsg1 antibodies quite difficult. The pathophysiology of the acantholytic phenomenon could depend on the reduction of Dsg1 adhesion function occurring after its massive internalization or decrease of its synthesis. Here, we have investigated this hypothesis by using sera of patients having antibodies against Dsg1 or monoclonal anti-Dsg1 antibodies to simulate pemphigus autoimmunity in Dsg1-rich keratinocytes. Similar to pemphigus foliaceus (PF) and vulgaris (PV) sera, monoclonal anti-Dsg1 antibodies induced transient internalization of Dsg1 and reduced the adhesion strength among keratinocytes. However, binding of IgG to Dsg1 did not determine its early depletion from the adhesion complexes but reduced the amount of Dsg1 found in the Triton X-100 soluble pool of proteins. Taken together, our results represent the first demonstration that anti-Dsg1 antibodies induce similar alterations on the subcellular distribution of Dsg1 irrespective of the disease where they come from. Furthermore, the present study provides insight into the mechanisms underlying epithelial blistering observed in the skin type of pemphigus.     

Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4⁺ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.     

Immune modulation in pemphigus vulgaris: role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28-/- mice keratinocytes. No alteration in in vitro adhesion was detected in CD28-/--type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28-/- mice. Lower levels of IL-10 mRNA were found in lesions from CD28-/- mice. To determine whether pemphigus susceptibility in CD28-/- was related to IL-10 deficiency, we performed passive transfer experiments in IL-10-/- mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.     

Th17 cell-derived IL-17 is dispensable for B cell antibody production

IL-17, which is preferentially produced by Th17 cells, is important for host defense against pathogens and is also involved in the development of autoimmune and allergic disorders. Antibody (Ab) production was shown to be impaired in IL-17-deficient mice, suggesting that IL-17 may promote B cell activation and direct secretion of Ab. However, the precise role of IL-17 in Ab production by B cells remains unclear. In the present study, we found constitutive expression of IL-17R in murine splenic B cells. Nevertheless, IL-17, IL-17F or IL-25 alone could not induce Ab production by B cells even in the presence of agonistic anti-CD40 Ab. IL-17 also could not affect IFN-γ-, IL-4- or TGF-β1-mediated Ig class-switching. Furthermore, in co-cultures of B cells and IL-17(-/-) CD4(+) T cells or IL-17(-/-) Th17 cells, IL-17 deficiency did not influence Ab production by B cells in vitro, suggesting that Th17 cell-derived IL-17 was not required for B cell Ab production through T cell-B cell interaction in vitro. Thus, in vivo, IL-17 may be indirectly involved in Ab production by enhancing production of B cell activator(s) by other immune cells.     

IFN-α confers resistance of systemic lupus erythematosus nephritis to therapy in NZB/W F1 mice

The critical role of IFN-α in the pathogenesis of human systemic lupus erythematosus has been highlighted in recent years. Exposure of young lupus-prone NZB/W F1 mice to IFN-α in vivo leads to an accelerated lupus phenotype that is dependent on T cells and is associated with elevated serum levels of BAFF, IL-6, and TNF-α, increased splenic expression of IL-6 and IL-21, formation of large germinal centers, and the generation of large numbers of short-lived plasma cells that produce IgG2a and IgG3 autoantibodies. In this study, we show that both IgG2a and IgG3 autoantibodies are pathogenic in IFN-α-accelerated lupus, and their production can be dissociated by using low-dose CTLA4-Ig. Only high-dose CTLA4-Ig attenuates both IgG2a and IgG3 autoantibody production and significantly delays death from lupus nephritis. In contrast, BAFF/APRIL blockade has no effect on germinal centers or the production of IgG anti-dsDNA Abs but, if given at the time of IFN-α challenge, delays the progression of lupus by attenuating systemic and renal inflammation. Temporary remission of nephritis induced by combination therapy with cyclophosphamide, anti-CD40L Ab, and CTLA4-Ig is associated with the abrogation of germinal centers and depletion of short-lived plasma cells, but relapse occurs more rapidly than in conventional NZB/W F1 mice. This study demonstrates that IFN-α renders NZB/W F1 relatively resistant to therapeutic intervention and suggests that the IFN signature should be considered when randomizing patients into groups and analyzing the results of human clinical trials in systemic lupus erythematosus.     

Leishmania amazonensis-dendritic cell interactions in vitro and the priming of parasite-specific CD4(+) T cells in vivo

The progressive disease following Leishmania amazonensis infection in mice requires functional CD4(+) T cells, which are primed to a disease-promoting phenotype during the infection. To understand how these pathogenic T cells are generated and the role of dendritic cells (DCs) in this process, we use DCs of susceptible BALB/c and resistant C3H/HeJ mice to examine parasite-DC interactions in vitro as well as the effector phenotype of T cells primed by parasite-exposed DCs in vivo. Our results demonstrate that amastigotes and metacyclics efficiently enter and activate DCs of both genetic backgrounds. Infection with amastigotes fails to induce CD40-dependent IL-12 production, but rather potentiates IL-4 production in BALB/c DCs. Upon transfer into syngeneic recipients, amastigote-exposed BALB/c DCs prime parasite-specific Th cells to produce significantly higher levels of IL-4 and IL-10 than their C3H/HeJ counterparts. Transfer studies with IL-4(-/-) DCs indicate that this enhanced Th2 priming seen in BALB/c mice is partially due to the IL-4 production by amastigote-carrying DCs. These results suggest that L. amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4(+) T cells, and thereby provide a new perspective on the pathogenesis of cutaneous leishmaniasis and protozoan parasite-host interactions in general.