Shu Man Fu

A Major Lung CD103 (αE)-β7 Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins

Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid organs. The third and largest DC population is the integrin αE (CD103) β7-positive and I-AhighCD11chigh-DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin αEβ7+ DC (αE-DC) were distinct from intraepithelial lymphocytes and distinguishable from CD11bhigh myeloid and mPDCA-1+B220+Gr-1+ PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, αE-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that αE-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis.

Breaking Tolerance to Double Stranded DNA, Nucleosome, and Other Nuclear Antigens Is Not Required for the Pathogenesis of Lupus Glomerulonephritis

In lupus-prone NZM2328 mice, a locus Cgnz1 on chromosome 1 was linked to chronic glomerulonephritis, severe proteinuria, and early mortality in females. A locus Adnz1 on chromosome 4 was linked to antinuclear antibody (ANA) and anti–double stranded DNA (dsDNA) antibody (Ab) production. In this investigation, two congenic strains, NZM2328.C57L/Jc1 (NZM.C57Lc1) and NZM2328.C57L/Jc4 (NZM.C57Lc4), were generated by replacing the respective genetic intervals containing either Cgnz1 or Adnz1 with those from C57L/J, a nonlupus-prone strain. The NZM.C57Lc1 females had markedly reduced incidence of chronic glomerulonephritis and severe proteinuria. NZM.C57Lc4 females had chronic glomerulonephritis and severe proteinuria without circulating ANA, anti-dsDNA, and antinucleosome Ab. These data confirm the linkage analysis. Unexpectedly, NZM.C57Lc1 females had little anti-dsDNA and related Ab, suggesting the presence of a second locus Adnz2 on chromosome 1. The diseased NZM.C57Lc4 kidneys had immune complexes by immunofluorescence and electron microscopy. The eluates from these kidneys did not contain ANA, anti-dsDNA, and antinucleosome Ab, indicative of the presence of non–anti-dsDNA nephritogenic Ab. Thus, breaking tolerance to dsDNA and chromatin is not required for the pathogenesis of lupus nephritis. These results reaffirm that anti-dsDNA and related Ab production and chronic glomerulonephritis are under independent genetic control. These findings have significant implications in the pathogenesis of systemic lupus erythematosus.

Pathogenesis of kidney disease in systemic lupus erythematosus

Purpose of reviewA combination of systemic autoimmunity and tissue response to immune injury underlie renal involvement in lupus erythematosus. In this review, we discuss recent literature investigating pathogenetic mechanisms of lupus glomerulonephritis. Recent findingsIn lupus glomerulonephritis, glomerular immune complexes were believed to be the primary mediators of renal disease. Recent studies make it apparent that autoantibodies of multiple specificities participate in the formation of immune complexes, deposited in the kidneys. Renal infiltration by T cells, macrophages, and dendritic cells have a dominant role in the progression of lupus glomerulonephritis leading to renal failure. Activation of Toll-like receptors modulates autoantibody production and systemic interferon responses. However, glomerular cell responses to immune injury influence disease outcome. In addition, new insights on the genetics of susceptibility to end-organ damage in lupus glomerulonephritis have been discovered. Differential glomerular responses reflected in gene expression profiles during disease progression provide potential markers for diagnosis of lupus glomerulonephritis progression and flares. In addition, studies of end-organ responses provide new targets for therapeutic interventions. SummaryLupus glomerulonephritis is a prototype of immune complex disease mediated by autoantibodies of multiple specificities, one of which is anti-DNA. Murine models of spontaneous systemic lupus erythematosus have been critical for understanding the underlying disease. Recent studies demonstrate that in addition to systemic autoimmunity, end-organ responses, and end-organ resistance to damage are also critical in determining disease outcome. This understanding should influence design of novel therapeutic approaches in systemic lupus erythematosus.

P2X7 Blockade Attenuates Murine Lupus Nephritis by Inhibiting Activation of the NLRP3/ASC/Caspase 1 Pathway

OBJECTIVE The NLRP3 inflammasome plays key roles in inflammation and autoimmunity, and purinergic receptor P2X7 has been proposed to be upstream of NLRP3 activation. The aim of the present study, using murine models, was to investigate whether the P2X7 /NLRP3 inflammasome pathway contributes to the pathogenesis of lupus nephritis (LN). METHODS MRL/lpr mice were treated with the selective P2X7 antagonist brilliant blue G (BBG) for 8 weeks. Following treatment, the severity of renal lesions, production of anti-double-stranded DNA (anti-dsDNA) antibodies, rate of survival, activation of the NLRP3/ASC/caspase 1 inflammasome pathway, and ratio of Th17 cells to Treg cells were evaluated. P2X7 -targeted small interfering RNA (siRNA) was also used for in vivo intervention. Similar evaluations were carried out in NZM2328 mice, a model of LN in which the disease was accelerated by administration of adenovirus-expressing interferon-α (AdIFNα). RESULTS Significant up-regulation of P2X7 /NLRP3 inflammasome signaling molecules was detected in the kidneys of MLR/lpr mice as compared with normal control mice. Blockade of P2X7 activation by BBG suppressed NLRP3/ASC/caspase 1 assembly and the subsequent release of interleukin-1β (IL-1β), resulting in a significant reduction in the severity of nephritis and circulating anti-dsDNA antibodies. The lifespan of the treated mice was significantly prolonged. BBG treatment reduced the serum levels of IL-1β and IL-17 and the Th17:Treg cell ratio. Similar results were obtained by specific siRNA silencing of P2X7 in vivo. The effectiveness of BBG treatment in modulating LN was confirmed in NZM2328 mice with AdIFNα-accelerated disease. CONCLUSION Activation of the P2X7 signaling pathway accelerates murine LN by activating the NLRP3/ASC/caspase 1 inflammasome, resulting in increased IL-1β production and enhanced Th17 cell polarization. Thus, targeting of the P2X7 /NLRP3 pathway should be considered as a novel therapeutic strategy in patients with lupus.

Intratracheal Priming with Ovalbumin- and Ovalbumin 323–339 Peptide-Pulsed Dendritic Cells Induces Airway Hyperresponsiveness, Lung Eosinophilia, Goblet Cell Hyperplasia, and Inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA323–339 and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA323–339-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.

Deficiency in regulatory T cells results in development of antimitochondrial antibodies and autoimmune cholangitis

There have been several descriptions of mouse models that manifest select immunological and clinical features of autoimmune cholangitis with similarities to primary biliary cirrhosis in humans. Some of these models require immunization with complete Freunds adjuvant, whereas others suggest that a decreased frequency of T regulatory cells (Tregs) facilitates spontaneous disease. We hypothesized that antimitochondrial antibodies (AMAs) and development of autoimmune cholangitis would be found in mice genetically deficient in components essential for the development and homeostasis of forkhead box 3 (Foxp3)+ Tregs. Therefore, we examined Scurfy (Sf) mice, animals that have a mutation in the gene encoding the Foxp3 transcription factor that results in a complete abolition of Foxp3+ Tregs. At 3 to 4 weeks of age, 100% of animals exhibit high‐titer serum AMA of all isotypes. Furthermore, mice have moderate to severe lymphocytic infiltrates surrounding portal areas with evidence of biliary duct damage, and dramatic elevation of cytokines in serum and messenger RNAs encoding cytokines in liver tissue, including tumor necrosis factor α, interferon‐γ, interleukin (IL)‐6, IL‐12, and IL‐23. Conclusion: The lack of functional Foxp3 is a major predisposing feature for loss of tolerance that leads to autoimmune cholangitis. These findings reflect on the importance of regulatory T cells in other murine models as well as in patients with primary biliary cirrhosis. (HEPATOLOGY 2008.)

Role for Nephritogenic T Cells in Lupus Glomerulonephritis: Progression to Renal Failure Is Accompanied by T Cell Activation and Expansion in Regional Lymph Nodes

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c+ dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vβ families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.

Extreme variation in X-linked agammaglobulinemia phenotype in a three-generation family

BACKGROUND X-linked agammaglobulinemia is typically a severe life-threatening disease characterized by the failure of B-cell differentiation and antibody production, which manifests in infancy and early childhood. Recently, we reported a novel mutation (Cys145-->STOP) in Brutons tyrosine kinase in a 51-year-old man who was referred for evaluation because of chronic nasal congestion, recurrent sinusitis, sporadic pneumonia, and a family history suggestive of an X-linked immunodeficiency disease. He had not been treated with gammaglobulin. OBJECTIVE This study was performed to investigate the clinical and immunologic phenotypes of this patients other affected male family members. METHODS A detailed family history and comprehensive review of medical records was carried out. Genetic mutation analysis of the gene encoding Brutons tyrosine kinase was carried out in the probands brother and nephew. RESULTS Clinically affected male family members exhibit marked phenotypic variation with manifestations ranging from extremely mild to severe recurrent infections. Immunologic evaluation revealed extreme variation in immunoglobulin levels, B-cell numbers, and functional antibody titers. Genetic analysis documented a novel mutation in the gene encoding Brutons tyrosine kinase in the proband, his brother, and his nephew. CONCLUSIONS Despite their sharing the same genetic abnormality, extreme variation was noted in the immunologic findings and phenotypic expression of affected family members. This family study is extraordinary in that clinically affected male members who did not receive aggressive medical treatment died of the disease in childhood or survived into late adulthood.

HLA Class II Influences the Immune Response and Antibody Diversification to Ro60/Sjögren’s Syndrome-A: Heightened Antibody Responses and Epitope Spreading in Mice Expressing HLA-DR molecules

Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sjögren’s syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61–185) and the carboxy portion (aa 381–525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281–315 and 401–538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.

Ro60 peptides induce antibodies to similar epitopes shared among lupus-related autoantigens.

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316–335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128–285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316–335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316–335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.