CD19 Expression in B Cells Regulates Atopic Dermatitis in a Mouse Model

Atopic dermatitis is an inflammatory cutaneous disorder characterized by dry skin and relapsing eczematous skin lesions. Besides antibody production, the contribution of B cells to the pathogenesis of atopic dermatitis is unclear. In mice, repeated epicutaneous sensitization with ovalbumin induces inflamed skin lesions resembling human atopic dermatitis and therefore serves as an experimental model for this condition. To investigate the role of B cells in a murine model of atopic dermatitis, ovalbumin-sensitized allergic skin inflammation was assessed in mice lacking CD19. In ovalbumin-sensitized skin from CD19-deficient mice, the number of eosinophils and CD4⁺ T cells was reduced, and both epidermal and dermal thickening were decreased. Following in vitro stimulation with ovalbumin, CD19 deficiency significantly reduced the proliferation of CD4⁺, but not CD8⁺, T cells from spleen and draining lymph nodes. Furthermore, splenocytes and draining lymph node cells from ovalbumin-sensitized CD19-deficient mice secreted significantly less IL-4, IL-13, and IL-17 than ovalbumin-sensitized wild-type mice. These results suggest that CD19 expression in B cells plays a critical role in antigen-specific CD4⁺ T-cell proliferation and T helper 2 and 17 responses in a murine model of atopic dermatitis. Furthermore, the present findings may have implications for B-cell–targeted therapies for the treatment of atopic dermatitis.Atopic dermatitis (AD) is one of the most common inflammatory cutaneous disorders, characterized by dry, itchy skin and relapsing eczematous skin lesions, which affects approximately 15% to 30% of children and 2% to 10% of adults.¹ Histologically, AD is characterized by epidermal and dermal thickening with marked infiltration of activated T cells, eosinophils, and monocytes/macrophages within the dermis.¹ Approximately 60% to 90% of patients with AD show increased serum total IgE against environmental and/or food allergens.^2–4 In addition, the expression of T helper (Th) 2 cytokines, such as IL-4, IL-5, and IL-13, is increased in the acute skin lesions of AD,^5,6 suggesting that Th2 cells play critical roles in disease development.Skin barrier dysfunction is a critical feature of AD. Recent studies have shown that more than 10% of patients with AD have mutations in the filaggrin gene, which is important for skin barrier function.^7,8 It has been hypothesized that a disrupted skin barrier facilitates antigen penetration and epicutaneous sensitization, leading to allergic skin inflammation in patients with AD.⁹ Moreover, IL-4 and IL-13 reduce filaggrin gene and protein expression in keratinocytes.¹⁰ Thus, a genetic and/or acquired defect in filaggrin is likely to play an important role in the development of AD. In mice, repeated epicutaneous sensitization of tape-stripped skin with ovalbumin (OVA), mimicking epicutaneous allergen exposure to epidermal barrier dysfunction, was found to induce the appearance of inflamed pruritic skin lesions at the application site, as well as local and systemic Th2 responses. Because of the resemblance of these lesions to human AD,^11,12 this experimental method can serve as a convenient experimental model.Historically, B cells have been considered to mediate humoral immune responses by differentiating into antibody (Ab)-secreting plasma cells.¹³ However, recent studies have revealed that B cells also serve as antigen-presenting cells,¹⁴ secrete a variety of cytokines,¹⁵ provide costimulatory signals, and promote T-cell activation.^15,16 Moreover, IL-10–producing B cell subsets can inhibit innate and adaptive immune responses, inflammation, and autoimmunity, demonstrating the existence of regulatory B cells.^13,17–19 Thus, in addition to Ab production, B cells have multiple diverse immune functions.The fate and function of B cells are controlled by signal transduction through B-cell receptors, which are further modified by other cell-surface molecules, including CD19, CD21, CD22, CD40, CD72, and Fcγ receptor IIb.²⁰ CD19 is a general rheostat that defines signaling thresholds critical for humoral immune responses and autoimmunity.²¹ CD19 is a B-cell–specific cell-surface molecule of the Ig superfamily expressed by early pre-B cells in humans and mice until plasma cell differentiation.^22,23 Human CD19 and mouse CD19 are functionally equivalent in vivo.²² B cells from CD19-deficient (CD19^−/−) mice are hyporesponsive to a variety of transmembrane signals, including B-cell receptor ligation.²² CD20, a B-cell–specific cell-surface molecule involved in the regulation of B-cell activation and Ca²⁺ transport, is initially expressed by pre-B cells in humans and mice with continued expression until plasma cell differentiation.^24,25 Although the role of B cells, besides Ab production, in the pathogenesis of AD remains unclear, B-cell depletion in humans with the chimeric human anti-CD20 monoclonal antibody (mAb) rituximab results in an improvement of AD,^26,27 suggesting that B cells play important roles in the development of this condition. Therefore, in the present study, we examined the importance of B cells in an OVA-sensitized allergic skin inflammation model using CD19^−/− and wild-type (WT) mice.     

Altered Peripheral Invariant Natural Killer T Cells in Atopic Dermatitis

Background  

Conflicting data exist on the number of invariant NKT (iNKT) cells in atopic dermatitis (AD); furthermore, no data have been published on their functional capacity.     

Upregulation of CD47 in Regulatory T Cells in Atopic Dermatitis

PurposeRegulatory T (Treg) cells are key modulators in the immune system. Recent studies have shown that atopic dermatitis (AD) patients have higher numbers of Treg cells; however, little is known about the specific phenotype and function of Treg cells in AD.ResultsUsing TMT labeling, we identified 510 proteins, including 63 membrane proteins and 16 plasma membrane proteins. CD47 was one of the upregulated proteins in Treg cells in AD spleens. Although CD47 was expressed in all CD4⁺ and CD8⁺ T cells, a significantly higher expression of CD47 was observed in the Treg cells of AD mice and AD patients than in those of normal mice and healthy controls. Furthermore, Treg cells from the spleen showed a significantly higher expression of CD47 than those from the thymus.ConclusionWe found that CD47 is highly expressed in the Treg cells of AD mice, particularly in the spleen. Based on our results, we propose that CD47^high Treg cells are likely induced Treg cells and that upregulated CD47 in the Treg cells of AD patients may play a role in the increased population of Treg cells in AD.     

Expression of Co-stimulatory Molecules on Langerhans Cells in Lesional Epidermis of Human Atopic Dermatitis

Langerhans cells (LC) are immature dendritic cells (DC) present in the skin epithelium. To understand the molecular and cellular mechanisms governing the inflammatory reaction in atopic dermatitis (AD), the expression of the LC specific marker CD1a, a member of major histocompatibility (MHC)-like glycoproteins, and the co-stimulatory molecules CD80 and CD86, expressed on functionally mature dendritic cells, were counted in lesional biopsies and normal epidermis by an immunohistochemical method. CD1a specific staining was observed in both normal and AD lesion specimens. CD80 and CD86 positive cells with morphological characteristics of the LC were found in lesional AD epidermis, suggesting a high level of functional maturity of these cells and their involvement in chronic inflammatory disease.     

Attenuation of Peripheral Regulatory T-Cell Suppression of Skin-Homing CD8+T Cells in Atopic Dermatitis

Purpose

Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8⁺T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8⁺CLA⁺T cells might be underlying mechanism in AD.

Materials and Methods

CD8⁺CLA⁺T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8⁺CLA⁺T cells were evaluated. The proliferative responses of CD8⁺CLA⁺T cells were assessed by flow cytometry, and the levels of transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay.

Results

Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8⁺CLA⁺T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8⁺CLA⁺T cells in AD. Meanwhile, the levels of TGF-β1 produced by Tregs were significantly lower in AD, and anti-TGF-β1 abolished such suppression.

Conclusion

The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8⁺CLA⁺T cells, mediated by TGF-β1, plays an important role in the pathogenesis of AD.     

Flaky Tail Mouse Denotes Human Atopic Dermatitis in the Steady State and by Topical Application with Dermatophagoides pteronyssinus Extract

The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. Flaky tail (Flg^ft) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin on AD. However, the relevancy of Flg^ft mice to human AD needs to be determined further. In this study, we observed the clinical manifestations of Flg^ft mice in the steady state and their cutaneous immune responses against external stimuli, favoring human AD. Under specific pathogen-free conditions, the majority of Flg^ft mice developed clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction evaluated by newly devised methods. In addition, cutaneous hapten-induced contact hypersensitivity as a model of acquired immune response and a mite extract-induced dermatitis model physiologically relevant to a human AD were enhanced in Flg^ft mice. These results suggest that the Flg^ft mouse genotype has potential as an animal model of AD corresponding with filaggrin mutation in human AD.Atopic dermatitis (AD), which affects at least 15% of children in developed countries, is characterized by eczematous skin lesions, dry skin, and pruritus.^1,2,3 Although the precise pathogenic mechanism of AD is as yet unknown, several accumulated lines of evidence suggest that a defective skin barrier to environmental stimuli may contribute to its pathogenesis. It has long been thought that the barrier abnormality in AD is not merely an epiphenomenon but rather is the “driver” of disease activity.⁴ The evidence for a primary structural abnormality of the stratum corneum in AD is derived from a recently discovered link between the incidence of AD and loss-of-function mutations in the gene encoding filaggrin (FLG). Individuals carrying the FLG null allele variants tend to develop AD.^5,6,7Filaggrin protein is localized in the granular layers of the epidermis. Profilaggrin, a 400-kDa polyprotein, is the main component of keratohyalin granules.^8,9,10 In the differentiation of keratinocytes, profilaggrin is dephosphorylated and cleaved into 10 to 12 essentially identical 27-kDa filaggrin molecules, which aggregate in the keratin cytoskeleton system to form a dense protein-lipid matrix.¹⁰ This structure is thought to prevent epidermal water loss and impede the entry of external stimuli, such as allergens, toxic chemicals, and infectious organisms. Therefore, filaggrin is a key protein in the terminal differentiation of the epidermis and in skin barrier function.¹¹Because AD is a common disease for which satisfactory therapies have not yet been established, understanding the mechanism of AD through animal models is an essential issue.^1,12 Flaky tail (Flg^ft) mice, first introduced in 1958, are spontaneously mutated mice with abnormally small ears, tail constriction, and a flaky appearance of the tail skin, which is most evident between 5 and 14 days of age.¹³ Mice of the Flg^ft genotype express an abnormal profilaggrin polypeptide that does not form normal keratohyalin F granules and is not proteolytically processed to filaggrin. Therefore, filaggrin is absent from the cornified layers in the epidermis of the Flg^ft mouse.^14,15,16Recently, it has been revealed that the gene responsible for the characteristic phenotype of Flg^ft mice is a nonsense mutation of 1-bp deletion analogous to a common human FLG mutation.¹⁵ These mice developed eczematous skin lesions after age 28 weeks under specific pathogen-free (SPF) conditions¹⁷ and enhanced penetration of tracer perfusion determined by ultrastructural visualization,¹⁶ and were predisposed to develop an allergen-specific immune response after epicutaneous sensitization with the foreign allergen ovalbumin (OVA).^15,17 On the other hand, general immunity through intraperitoneal sensitization with OVA was comparable between Flg^ft mice and control mice.^15,17Despite these recent advances, there still remain several issues with Flg^ft mice to be addressed. For example, serial close observation of clinical manifestations in reference to human AD will be informative. It is of value to evaluate the responses to external stimuli relevant to human AD, such as mite extracts, instead of OVA that has been used previously. A comparative study on the skin-mediated contact hypersensitivity (CHS) response and non-skin-mediated delayed-type hypersensitivity response is important to evaluate the impact of barrier dysfunction on immune responses in vivo. In addition, although it has now been determined that the barrier dysfunction is a key element in the establishment of AD, there is no established method to evaluate the outside-to-inside barrier function quantitatively.In this study, we found that Flg^ft mice showed spontaneous dermatitis with skin lesions mimicking human AD in a steady state under SPF conditions: serial occurrence of manifestations as scaling, erythema, pruritus, and erosion followed by edema in this order. We also successfully evaluated outside-to-inside barrier dysfunction in Flg^ft mice quantitatively using a newly developed method. In addition, we determined that the Th1/Tc1-mediated immune response was enhanced by immunization through skin but not through non-skin immunization. Last, we induced severe AD-like skin lesions in Flg^ft mice by application of mites as a physiologically relevant antigen for human AD, which will be an applicable animal model of AD.     

Human γδ T Cells Induce Dendritic Cell Maturation

γδ T cells are known to be involved in the innate immune defenses against infectious microorganisms. Herein, we considered that γδ T cells could also influence adaptative immunity by interacting with dendritic cells (DC) in the early phase of the immune response. To investigate this hypothesis, γδ T cells isolated from the peripheral blood of healthy volunteers were cocultured with autologous monocyte-derived dendritic cells, which were subsequently analyzed for their expression of key surface molecules and for their production of IL-12. First, we found that γδ T cells induced the upregulation of HLA-DR, CD86, and CD83 on DC. This effect did not require cell to cell contact and could be blocked by a neutralizing anti-TNF antibody. We then observed that γδT cells activated by the synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) induced the production of IL-12 (p40) and IL-12 (p70) by DC, an effect that involved IFN-γ production. The relevance of this finding to DC function was demonstrated by the increased production of IFN-γ by alloreactive T cells when stimulated in a mixed leucocyte reaction with DC preincubated with activated γδ T cells. We conclude that γδ T cell activation might result in DC maturation and thereby in enhanced αβ T cell responses.     

Murine and Human T Cell Factors that Induce the Differentiation of Normal Mouse Lymphocytes into Cytotoxic Cells Copurify with Interleukin 2

Fetal calf serum (FCS)-specific T promoter cell lines (line 12), clones, or lymphomas produce lymphocyte promoter factors (LPF). These factors are defined as T-cell supernatant activities that induce polyclonal differentiation of normal experimentally unprimed mouse lymphocytes into antibody-forming cells (B-LPF) or into cytotoxic cells (T-LPF). The cytotoxic cells thus induced lysed a broad range of target cells including syngeneic and allogeneic tumour cells and lymphoblasts. We have investigated whether T cell tumours (mouse or human) other than FCS-specific T promoter cell lines (line 12), clones, or lymphomas produce T-LPF activity, and whether T-LPF activity is related to interleukin 2 (IL-2) activity. We found that the EL4 thymoma cells were high producers of T-LPF and IL-2 activity. When EL4 cells and T-LPF ⁺ line 12 lymphomas were cloned, all T-LPF high-producer clones were also high IL-2 producers. In addition, the human Jurkat T tumour cells produced both T-LPF and IL-2 activity which could he detected on both mouse and human lymphocytes. By using biochemical fractionation (size fractionation or chromatofocusing fractionation) and absorption techniques, we could not separate T-LPF and IL-2 activity. Thus, the present data may indicate that the T-LPF and IL-2 activities studied in the present systems are borne by the same molceule(s) (=IL-2?). These results are discussed in relation to current hypotheses on the cellular and molecular requirements for the generation of cytotoxic T cells.     

Natural Killer Cells and Cytotoxic T Cells Induce DNA Fragmentation in Both Human and Murine Target Cells in Vitro

DNA fragmentation induced by cytolytic lymphocytes in human erythromyeloid cell line K562 and murine T lymphoma cell line YAC-1 was investigated by means of agarose gel electrophoresis. Murine natural killer (NK) and cytotoxic T (Tc) cells induced DNA fragmentation in YAC-1 cells, with the fragments being approximately multiples of 180 bp. More significantly, murine NK cells can induce a similar pattern of DNA fragmentation in human K562 cells. Therefore, cytolytic lymphocytes can induce apoptosis or programmed cell death in human target cells.     

A stromal cell free culture system generates mouse pro‐T cells that can reconstitute T‐cell compartments in vivo

T‐cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage^?Sca1⁺c‐kit⁺ BM hematopoietic progenitors very efficiently differentiate into pro‐T cells. This culture system consists of plate‐bound Delta‐like 4 Notch ligand and the cytokines SCF and IL‐7. The pro‐T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D–J rearranged. They could be expanded for over 3 months and used to reconstitute the T‐cell compartments of sublethally irradiated T‐cell‐deficient CD3ε^?/? mice or lethally irradiated WT mice. Pro‐T cells generated in this system could partially correct the T‐cell lymphopenia of pre‐Tα^?/? mice. However, reconstituted CD3ε^?/? mice suffered from a wasting disease that was prevented by co‐injection of purified CD4⁺ CD25^high WT Treg cells. In a T‐cell‐sufficient or T‐lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro‐T cells for transplantation and possibly with clinical applications.     

Antigen‐presenting Cells in Pregnant and Non‐pregnant Human Myometrium

Citation Ivanisevic M, Segerer S, Rieger L, Kapp M, Dietl J, Kämmerer U, Frambach T. Antigen‐presenting cells in pregnant and non‐pregnant human myometrium. Am J Reprod Immunol 2010; 64: 188–196 Problem Inflammatory cells play a crucial role in human parturition. Different populations of leucocytes invade the reproductive tract. Numerous studies have described the decidual immune cell population in pregnant and non‐pregnant endometrium. However, little is known about the presence of immune cells in human myometrium. Method of study We herein analysed a spectrum of immune cells in human myometrium comparing tissue samples from non‐pregnant (n = 8) and pregnant (n = 10) uteri. Applying immunohistochemistry with a panel of antibodies specific for T cells, monocytes, natural killer cells, B cells and antigen‐presenting cells (CD4, CD8, CD14, CD15, CD16, CD19, CD56, CD68, CD83, HLA‐DR, DC‐Sign, mast cell tryptase), we characterized the immune cell population of human myometrium. Results A significantly higher number of CD14, CD15, CD16, DC‐SIGN as well as CD4‐positive cells were found in myometrium of pregnant compared to non‐pregnant uteri, while mast cells were significantly reduced in pregnant myometrium. Conclusion All markers found increased in pregnant myometrium indicate monocyte/macrophage lineage cells and thus suggest a possible involvement of these cells in healthy pregnancy maintenance. Monocytes/macrophages might produce a microenvironment that permits a controlled invasion of trophoblast cells into the myometrium while preventing a rejection of the semiallogenic conceptus and providing an important barrier against invading pathogenes.     

Fc Receptors on Human T Lymphocytes: Loss of Fcμ and Expression of Fcγ Receptors by T Cells Stimulated in Mixed Lymphocyte Reaction

The consequences of allogeneic stimulation on the expression of Fc mu or Fc gamma receptors were analysed in T-cell populations responding in mixed lymphocyte reaction (MLR). Unfractionated T-cell populations were cultured with a pool or irradiated allogeneic T-depleted cells. The responder E-rosetting cells progressively lost Fc mu and acquired Fc gamma receptors. The change of the original Fc receptor phenotype is not the consequence of a preferential proliferative response of TG versus TM cells but is likely due to a de novo expression of Fc gamma receptors by T cells lacking detectable Fc receptors (T-null) and also to the loss of Fc mu and expression of Fc gamma receptors by TM cells. These data suggest that, after MLR, responder T cells can modify their Fc receptor phenotype.     

SEF14 fimbriae from Salmonella enteritidis play a role in pathogenitic to cell model in vitro and host in vivo

The role of SEF14 fimbriae in virulence remains to be elucidated and in this study, we showed that sefA mutant constructed in the wild-type (WT) Salmonella enteritidis strain 50336 displayed increased invasion to IPEC-J2 cell lines and survival in mouse peritoneal macrophages, and the lethal dose 50% (LD₅₀) in 6-week-old Balb/c mice intra-peritoneally injected with WT S. enteritidis strain decreased significantly upon deletion of sefA indicating their role in virulence. Overall, these results demonstrated that expression of sefA of SEF14 fimbriae enhances S. enteritidis adhesion to epithelial cells and survival in macrophages and contributes to S. enteritidis virulence in mice.