β-溶血型链球菌诱发银屑病发病机理探讨

探讨β-溶血性链球菌诱发银屑病的机制。采用3H-TdR掺入法测定细胞增殖反应,AnnexinV法测定细胞凋亡,流式细胞仪检测细胞表面抗原表达。结果显示β-溶血性链球菌(SP)刺激淋巴细胞活化增殖,经SP活化的淋巴细胞培养上清液作用于角质形成细胞48h,可促进角质形成细胞增殖,诱导角质形成细胞表达HLA-DR和Fas抗原;再次加入上清液继续作用48h,则诱导角质形成细胞凋亡。SP作为超抗原首先活化T细胞,使之释放细胞因子,后者使角质形成细胞活化增殖,表达和抗原,继而诱导细胞凋亡,此过程可能是银屑病的主要发病机制之一。     

链球菌抗原刺激的银屑病外周血单个核细胞培养上清液对角质形成细胞的作用

目的 了解链球菌抗原刺激的银屑病外周血单个核细胞(PBMCs)培养上清液对角质形成细胞的作用以探讨链球菌感染后的银屑病的发病机制。方法 ^3H—TdR掺入法检测PBMCs培养上清液对角质形成细胞DNA合成的影响；免疫组织化学方法检测细胞间教附因子1(ICAM—1)和人类白细胞抗原DR分子(HLA—DR)表达。结果 银屑病患者链球菌抗原刺激的PBMCs培养上清液对角质形成细胞的促增殖作用较对照组显著增强，并能诱导角质形成细胞表达HLIA—DR和ICAM—1分子。结论 受链球菌抗原刺激的银屑病PBMCs培养上清液可促进角质形成细胞增殖和活化，可能是链球菌感染之后银屑病发病的重要原因。     

HLA-DR expression by hair follicle keratinocytes in alopecia areata: evidence that it is secondary to the lymphoid infiltration

There is evidence suggesting that alopecia areata (AA) may have an autoimmune pathogenesis, and it was recently reported that keratinocytes in the bulb of some hair follicles affected by this condition express class II HLA (HLA-DR) antigens, which are not present on the same cells in normal tissue. Since it has been proposed that an analogous ectopic HLA-DR expression by epithelial cells in other organs might be an early event leading to organ-specific autoimmunity, we have investigated the sequence in which perifollicular mononuclear cell (MNC) infiltration and ectopic HLA-DR expression on keratinocytes appear in recent-onset and long-standing cases of AA by immunostainings of affected and unaffected areas with monoclonal antibodies against leukocyte and HLA-DR antigens. In recent-onset AA lesions, ectopic HLA-DR expression on hair follicle keratinocytes was found only occasionally (in 3 out of 247 follicles examined) and was restricted to biopsies from the affected areas. This prevalence was significantly lower than the prevalence of hair follicles showing perifollicular MNC infiltrates in the same biopsies, and was also significantly lower than the prevalence of hair follicles showing ectopic HLA-DR expression on keratinocytes in the affected areas of longstanding cases. These findings suggest that in AA lesions the perifollicular MNC infiltration precedes the ectopic HLA-DR expression on hair follicle keratinocytes, and therefore argue against the notion of a primary role for that ectopic HLA-DR expression on epithelial cells in triggering the putative autoimmune response in AA.     

Differential expression of Ia by murine keratinocytes and gut epithelium in response to recombinant gamma-interferon

Keratinocyte expression of class II antigens (HLA-DR, human; Ia, murine) is associated with certain cutaneous diseases, especially those marked by the infiltration of immune and inflammatory cells into the skin. It has been shown that interferon-gamma (IFN-gamma) is capable of inducing human keratinocytes to express HLA-DR. Similar results, however, have not been duplicated in murine systems. The purpose of this study was to determine whether IFN-gamma was capable of inducing murine keratinocyte expression of Ia in vivo in an experimental model in which epithelial cells in a variety of organs were shown to express Ia after the i.v. injection of IFN-gamma. Recombinant murine IFN-gamma was injected into BALB/c mice. Biopsies of skin and intestine were analyzed by indirect immunoperoxidase to identify Ia-expressing keratinocytes and mucosal cells, respectively. Interferon-gamma was administered as either: 1) a single s.c. injection, 2) multiple i.v. injections of increasing doses (10(3)-10(5) U/d) on 3 consecutive d, or 3) i.p. injections of 5 X 10(4) U/d or 5 X 10(5) U/d on 6 consecutive d. At all i.v. and i.p. injection doses, the intestinal villi mucosal cells were induced to express Ia. Keratinocyte expression of Ia, however, was observed only in animals that received the two higher i.p. doses. Procedures to augment Ia expression, e.g., combined treatment with pertussis toxin, dinitrofluorobenzene, tumor necrosis factor, and indomethacin, did not enhance the ability of IFN-gamma to induce keratinocyte expression of Ia. We conclude that: 1) high doses of IFN-gamma are required to induce murine keratinocyte Ia expression in vivo and 2) low doses of IFN-gamma, although capable of inducing intestinal mucosal cells to express Ia, do not induce keratinocyte Ia expression.     

金黄色葡萄球菌性超抗原诱发银屑病发病机理探讨

目的探讨金黄色葡萄球菌性超抗原诱发银屑病的机制。方法3H-TdR掺入法测定细胞增殖反应,亚二倍体细胞含量测定,片段化DNA分析,膜联蛋白V(AnnexinV)法测定细胞凋亡,流式细胞仪检测细胞表面抗原表达。结果金黄色葡萄球菌肠毒素B活化的淋巴细胞培养上清液作用于角质形成细胞48h,可促进角质形成细胞增殖,诱导角质形成细胞表达HLA-DR和Fas抗原;再次加入上清液继续作用48h,则诱导角质形成细胞凋亡（P<0.01）。结论金黄色葡萄球菌肠毒素B作为超抗原活化T细胞,使之释放细胞因子,后者使角质形成细胞首先活化增殖,继而凋亡。     

Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor

Lymphocytes bind to cultured keratinocytes that are treated with interferon gamma (IFN-gamma) and tumor necrosis factor (TNF). When the lymphocytes are preincubated with antibody to lymphocyte function associated antigen-1 (LFA-1), this adherence is inhibited. Because intercellular adhesion molecule-1 (ICAM-1) is a ligand for LFA-1, we studied the cellular expression of ICAM-1, as well as two other IFN-gamma-inducible antigens, (HLA) human lymphocyte antigens DR and DQ, in both normal and diseased skin. The modulation of these cell surface antigens by IFN-gamma and TNF with the use of short-term organ cultures of skin was compared with isolated keratinocytes grown in a conventional tissue culture system. While in normal skin, keratinocytes did not express HLA-DR, DQ, or ICAM-1, when organ cultures were supplemented with IFN-gamma, rapid induction of keratinocyte ICAM-1 expression occurred after 24 hours; HLA-DR but not DQ expression occurred after 48 hours. TNF also induced keratinocyte ICAM-1 expression (although to a lesser degree than IFN-gamma) but did not induce either keratinocyte HLA-DR or DQ expression. There was good correlation of keratinocyte expression of ICAM-1 and HLA-DR by IFN-gamma and TNF when the epidermis of the organ culture system was compared with the isolated keratinocytes grown in tissue culture. The presence of intraepidermal lymphocytes correlated extremely well with keratinocyte ICAM-1 expression but not with keratinocyte HLA-DR expression in psoriasis, atopic dermatitis, lichen planus, and mycosis fungoides. The intensity of endothelial cell expression of ICAM-1 correlated with the degree of dermal inflammation. We conclude that IFN-gamma, once produced by activated T lymphocytes in the dermis, may be of importance in lymphocyte trafficking in the epidermis by the induction of keratinocyte ICAM-1 expression. The use of the short-term organ culture system, in which there is inducible ICAM-1 expression, provides an experimental bridge between purely in vitro and in vivo investigations to further our understanding of the molecular basis for lymphocyte apposition to keratinocytes in the skin.     

Dynamics of Langerhans cells in genetically defined murine epidermal cell culture

Unlike keratinocytes, Langerhans cells express both surface ATPase activity and Ia (HLA-DR) antigens. A well-characterized in vitro system containing Langerhans cells would be of great use in elucidating their functions. Thus, epidermal cell cultures derived from neonatal Balb/c mice were examined for the presence of Langerhans cells. Twenty-four hours after initiation of culture, ATPase- and Ia-positive cells were seen to be associated with cell aggregates. By day 3, Langerhans cells migrated on to the substratum and, as the cultures matured and stratified, were seen both in groups and as single cells for the duration of the cultures (day 14). During culture, although the total number of cells increased, the percentage of cells expressing Ia antigen and ATPase activity remained constant, suggesting that Langerhans cells increase in number during cell culture. Such a situation could arise from actual division of Langerhans cells during culture or from latent expression of Ia antigen and ATPase activity by pre-existing cells. This is the first study of the dynamics of Langerhans cells in a cell culture system and shows that Langerhans cells are present throughout the lifespan of the cultures.     

Expression of OKM5 antigen on human keratinocytes in vitro upon stimulation with gamma-interferon

Using murine monoclonal antibodies against human OKM5, OKM1 and HLA-DR antigens antigenic characteristics of freshly separated human epidermal cells (EC) and those of EC cultured in the presence of Interferon-gamma (IFN-gamma) were studied. After 8-12 days of culture, primarily OKM1- OKM5- HLA-DR- keratinocytes displayed OKM5 and HLA-DR antigens when exposed to IFN-gamma. Our data support the concept, that human keratinocytes may possess accessory cell functions.     

The expression of MHC class II (Ia) antigens on mouse keratinocytes following epicutaneous application of contact sensitizers and irritants

The expression of MHC class II (Ia) antigens on mouse keratinocytes was studied following both the induction and elicitation of contact sensitivity, and after primary irritant reactions. IA+ and IE+ keratinocytes were detected, using an indirect immunofluorescence assay on epidermal sheets, only after the induction and elicitation of contact sensitivity with the sensitizers oxazalone, picryl chloride and 2,4-dinitrochlorobenzene but not with formaldehyde. Ia+ keratinocytes were not detected after epicutaneous application of the non-sensitizing irritants croton oil, SDS and anthralin, or following attempted sensitization of nude mice, suggesting that the expression of Ia antigen on keratinocytes during contact sensitivity reactions is T-cell mediated. Because Ia antigen expression on keratinocytes could be detected only several days after induction or elicitation of contact sensitivity, and contact sensitization could also be demonstrated to occur independently of aberrant Ia expression, Ia+ keratinocytes cannot be involved in the initiation of these reactions. However, they might be important in exerting an immunomodulatory influence during the later stages of the responses to certain sensitizers.     

Differential expression of major histocompatibility complex class II antigens on human keratinocytes

Skin biopsies from 136 patients with 30 different dermatoses and eight biopsies of normal skin were investigated with the avidin-biotin-peroxidase complex method with regard to the expression of major histocompatibility complex class II antigens human leukocyte antigen (HLA) HLA-DR, HLA-DQ, HLA-DP on keratinocytes. In normal skin the expression of these antigens was restricted to acrosyringia and Langerhans cells. In the dermatoses investigated HLA-DR was found in 51.5% (70 of 136), HLA-DQ in 24.3% (33 of 136), and HLA-DP in 20.5% (8 of 39). In 37 cases (27.2%) only HLA-DR could be detected, whereas in 33 cases (24.3%) HLA-DR was expressed jointly with HLA-DQ. Coexpression of HLA-DR and HLA-DQ was found especially often in cutaneous T cell lymphomas, skin tumors, and inflammatory dermatoses.     

Differential expression of class II alloantigens by keratinocytes in disease

The purpose of this study was to determine whether keratinocytes in certain disease states such as cutaneous T-cell lymphoma and lichen planus, express HLA-DR antigens (corresponding to the murine I-E antigens) only or whether they are also capable of expressing HLA-DQ antigens (analogues of the murine I-A antigens). Cryostat sections from 11 biopsies from cutaneous T-cell lymphoma and from 11 lichen planus biopsy specimens were submitted to indirect immunofluorescence and a 4-step immunoperoxidase method. This consists of applying monoclonal antibodies recognizing HLA-DR and HLA-DQ molecules and the intracytoplasmic invariant chain of the class II molecules. In 8 of the 11 cutaneous T-cell lymphoma specimens and in 3 of the 11 lichen planus biopsies concomitant expression of HLA-DR and HLA-DQ molecules by keratinocytes was detectable with the immunoperoxidase method. However, with the indirect immunofluorescence technique HLA-DQ antigens on keratinocytes could not be detected. The simultaneous expression of surface-bound HLA-DR antigens and intracytoplasmic gamma-chains was demonstrable in all cases investigated and with both the immunohistologic methods applied.     

Ultrastructural documentation of HLA-DR antigen reactivity in normal human acrosyringial epithelium

We have observed that monoclonal antibodies directed against human Ia-like antigens react with a subset of epidermal keratinocytes as well as Langerhans cells in normal human skin. Membrane reactivity for HLA-DR antigen in flattened ductal keratinocytes and in adjacent cuticular cells forming the acrosyringial lumen was observed using immunoelectron microscopy. It should be recognized that Ia-positive acrosyringial keratinocytes represent a potential source of contamination in methods designed to study and isolate Langerhans cells using antibodies directed against HLA-DR antigens. Teleologic considerations of HLA-DR antigen expression in the acrosyringium are discussed.     

Functional analysis of Ia antigen-bearing keratinocytes: mixed skin lymphocyte culture between Ia antigen-bearing Pam 212 cells and allogeneic and syngeneic splenic T cells

Keratinocytes express Ia antigens in various skin disorders, although the biological role of these Ia antigen-bearing (Ia+) keratinocytes remains unclear. We induced Ia antigens on Pam 212 murine keratinocyte cell line by interferon-gamma(IFN-gamma) and using these cells, we performed the mixed skin lymphocyte culture with syngeneic BALB/c or allogeneic C3H/He splenic T cells. Unexpectedly, Pam 212 cells were found to stimulate both syngeneic and allogeneic T cells irrespective of IFN-gamma treatment. However, both syngeneic and allogeneic T cells cultured with IFN-gamma-treated Pam 212 cells incorporated [3H]thymidine much more actively than those cultured with IFN-gamma-untreated Pam 212 cells. This stimulation was not inhibited by monoclonal anti-I-Ad antibody. Analysis of the responding T cells demonstrated that the syngeneic T-cell stimulation by IFN-gamma-treated Pam 212 cells occurred in both purified Lyt 1-T cells and Lyt 2- T cells. Furthermore, we found that the T cells cultured with the IFN-gamma-treated cells were composed of two morphologically different types of cells. Determination of their surface phenotype showed that the small cell population consisted of 57% Thy-1+, 23% Lyt-1+, 6% Lyt-2+, and 9% asialo-GM1+ cells, while the large cells consisted of 53% Thy-1+, 15% Lyt-1+, 9% Lyt-2+, and 24% asialo-GM1+ cells. These findings suggest that IFN-gamma-treated Pam 212 cells could stimulate more than one kind of splenic T cell populations.     

Alopecia areata: autoimmunity--the evidence is compelling

There is strong evidence indicating that alopecia areata is a tissue-specific, autoimmune disease. Hair loss is associated with a perifollicular lymphocytic infiltrate made up primarily of CD4+ cells, along with a CD8+ intrafollicular infiltrate. Evidence of immune activation includes expression of HLA-DR; HLA-A,B,C; and ICAM-1 on the follicular epithelium. It is likely that the follicular expression of HLA-DR and ICAM-1 is induced by interferon-gamma produced by T cells. Antibodies to follicular epithelium are often present, but their significance is not known. Lesional scalp from alopecia areata patients grafted onto nude mice regrows hair coincident with a loss of infiltrating lymphocytes from the graft. Hair loss can be transferred to human scalp explants on SCID mice by injection of lesional T cells. It is necessary to activate the T cells by culture with follicular autoantigens. Melanocyte-associated antigens are also capable of activating T cells to induce hair loss, suggesting that they are capable of functioning as autoantigens for alopecia areata. Parallel evidence in rodent models of spontaneous alopecia areata also strongly supports a role for T cells in the pathogenesis of this condition.     

Fungal infections inducing HLA-DR but not HLA-DQ transplantation antigens on keratinocytes

The phenotypes of infiltrating cells and class II transplantation antigens on keratinocytes in candida and dermatophyte lesions from 15 patients were analysed in situ with an immunohistochemical double staining technique combined with periodic acid-Schiff staining. In five out of ten biopsies from candida lesions and in one of five biopsies from dermatophyte lesions the keratinocytes expressed HLA-DR but not HLA-DQ antigens. The HLA-DR expression was patchy in all and most pronounced in two candida biopsies which also contained large infiltrates of anti-Leu 3a reactive T lymphocytes. The induction of detectable amounts of different class II antigens on keratinocytes might depend on the type of antigen, the magnitude and duration of the response elicited and/or the immunological state of the patient.     

Induction of Intercellular Adhesion Molecule-1 and Adherence of HTLV-1-Infected T-cells to Cultured Keratinocytes

Cutaneous lesions of T-cell proliferative disorders are characterized by epidermotropic infiltration of the neoplastic cells and expression of intercellular adhesion molecule-1 (ICAM-1) and HLA-DR by lesional keratinocytes. Using cloned HTLV-1-infected T-cells obtained from patients with adult T-cell leukemia (ATL), we have studied immunobiological activities of cytokines released from the T-cell lines and their ability to adhere to cultured keratinocytes. Three out of the five CD-4-positive, HTLV-1-infected T-cell clones secreted both IFN-γ and IL-4, similar to murine Th0 clones. The other two clones did not produce such cytokines. ICAM-1 and HLA-DR molecules were induced on cultured normal human keratinocytes and organ-cultured skin specimens by co-cultivation with IFN-γ-producing T-cell clones or their culture supernatants. Induction of both molecules was markedly inhibited by pretreatment of the supernatants with excess amounts of anti-IFN-γ monoclonal antibody. The number of cells adherent to the normal cultured keratinocytes was greater in the IFN-γ-producing clones than in the non-producing ones. These data suggest that some HTLV-1-infected clones produce cytokines, including IFN-γ, which in turn induce ICAM-1 on keratinocytes, thereby enhancing the ability of the T-cell clones to adhere to the keratinocytes.     

The cellular origins of the linear IgA disease target antigens: an indirect immunofluorescence study using cultured human keratinocytes and fibroblasts

BACKGROUND: Linear IgA disease (LAD) is an IgA-mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology. Objectives To investigate to what extent the cellular origins of the target antigens account for the heterogeneity of the immune response in LAD. METHODS: Forty-nine adult and 33 childhood LAD sera were studied. Immunofluorescence was carried out to determine the expression of the LAD antigens by normal human keratinocytes, fibroblasts and mixed cultures of keratinocytes and fibroblasts. Immunoblotting was performed to determine the localization of the LAD target antigens in tissue extracts (48 adult and 31 childhood sera) and cell extracts (21 adult and 10 childhood sera). RESULTS: Thirty-one adult and 13 childhood LAD sera bound proteins expressed by human keratinocytes; of these sera, 15 adult and four childhood LAD sera also recognized proteins expressed by fibroblasts. A single adult serum was positive on fibroblasts alone. Seventeen adult and 20 childhood sera were negative on both cell types. There was a modest increase (9%) in the detection of the IgA autoantibodies on keratinocytes and fibroblasts grown together in mixed culture. Immunoblotting showed that the LAD target antigens could be detected in cell as well as in tissue extracts. CONCLUSIONS: Our results have shown that normal human keratinocytes and fibroblasts in culture express the LAD target antigens. LAD sera (with a single exception) bound antigens expressed by keratinocytes alone or by both keratinocytes and fibroblasts. The principal pattern of expression in keratinocytes was cytoplasmic, similar to that demonstrated by polyclonal antibodies to the 180-kDa bullous pemphigoid antigen (BP180). This reflects the pivotal role of BP180 in LAD. The finding that LAD antigens are expressed by both human keratinocytes and fibroblasts in culture may explain the heterogeneity of the target antigens, and may be a contributory factor in the immunopathology of the disease.     

Recombinant gamma interferon and in vivo induction of HLA-DR antigens

Recombinant human IFN-gamma, used for treatment of melanoma and renal carcinoma, was found to induce HLA-DR expression on human keratinocytes in vivo. HLA-DR antigens bound to keratinocytes of the basal and suprabasal layers of the epidermis were observed after intramuscular or intravenous injections of 0.5 mg/kg body weight IFN-gamma, 3 times a week. Keratinocyte-bound HLA-DR antigens were first observed at the beginning of the third or fourth week of treatment, but HLA-DQ and HLA-DP antigens were never detected on keratinocytes. The intracytoplasmic constant (gamma) chain of the class II molecules was also not detectable within the keratinocytes. Patients who received IFN-alpha 2 therapy, did not exhibit keratinocyte-bound HLA-DR antigens.     

Gamma interferon induces different keratinocyte cellular patterns of expression of HLA-DR and DQ and intercellular adhesion molecule-I (ICAM-I) antigens

With indirect immunofluorescence techniques we demonstrated that recombinant gamma-interferon induced the expression of the class II antigens HLA-DR and HLA-DQ as well as intercellular adhesion molecule-I (ICAM-I) on normal, cultured human keratinocytes grown in low-calcium, serum-free medium. Each antigen displayed a distinctive cellular staining pattern. HLA-DR was strongly localized to perinuclear zones with intense cell surface expression; HLA-DQ displayed a perinuclear accentuation, but with minimal cell surface staining, and ICAM-I was strongly expressed in a diffuse cytoplasmic pattern with intense cell surface expression. Keratinocytes grown in medium supplemented with 10% fetal calf serum underwent differentiation, with a diminished expression of all three antigens as compared to those grown in low-calcium, serum-free medium. These results confirm that gamma interferon can differentially regulate HLA-DR and HLA-DQ expression; that there are probably different biochemical metabolic pathways by which these three molecules are expressed on keratinocytes, and that the expression is also a function of the degree of keratinocyte differentiation. The strong cell surface expression of ICAM-I is suggested to be of major importance as the recognition molecule, by which T cells bind to gamma interferon exposed keratinocytes, and suggests an integral role for this molecule in epidermal lymphocyte trafficking.