Recombinant Gamma Interferon Increases the Binding of Peripheral Blood Mononuclear Leukocytes and a Leu-3+ T Lymphocyte Clone to Cultured Keratinocytes and to a Malignant Cutaneous Squamous Carcinoma Cell Line That Is Blocked by Antibody Against the LFA-1 Molecule
Because keratinocytes (KCs) express HLA-DR in a wide variety of skin diseases in which mononuclear leukocytes are observed in close apposition to KCs (i.e., graft-versus-host disease), and since gamma interferon (IFN-gamma) induces HLA-DR expression on KCs, we asked whether IFN-gamma treatment of KCs would influence the adherence of mononuclear leukocytes. When allogeneic peripheral blood mononuclear leukocytes (PBML) and a Leu-3+ T cell clone were coincubated with IFN-gamma-treated KCs (300 U/ml, 3 days), there was a marked increase in binding compared with nontreated KCs. Similar binding results were obtained using a cutaneous squamous carcinoma cell line (SCL-1) after IFN-gamma treatment. The IFN effect was relatively specific for IFN-gamma, as neither IFN-alpha nor -beta had any effect. Tumor necrosis factor exposure (500 U/ml, 3 days) increased the binding of the Leu-3+ T cell clone to both KCs and SCL-1 cells. Neutrophils displayed a less marked (but statistically significant) increase in binding to IFN-gamma-treated KCs. Using the Leu-3+ cell clone and SCL-1 cells, detailed kinetic analysis of the effect of IFN-gamma on binding was performed. The increased adherence between the cells began to appear after only 7 hours of treatment with r-IFN-gamma (300 U/ml) and reached a plateau at 48 hours, with significantly enhanced binding continuing for at least 48 hours after removal of IFN-gamma. The mechanism of binding was explored by preincubation of the PBML/Leu-3+ T cells with anti-LFA-1 (lymphocyte function-associated antigen) antibody (0.6-6.0 micrograms/ml), which totally inhibited the binding with no effect by anti-LFA-2 or -3 or class I or II antibodies despite documented binding of these antibodies to the cells. These results suggest that, after exposure to IFN-gamma, the ability of KCs to bind mononuclear leukocytes is strongly enhanced, and this adherence may be important in leukocyte trafficking in the skin as well as contributing to altered KC-leukocyte interaction, which may be of fundamental importance in a variety of skin disease.
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[Show abstract][Hide abstract] ABSTRACT: Recombinant gamma interferon (r-IFN-gamma) increases the adherence of peripheral blood mononuclear leukocytes (PBMLs) to cultured keratinocytes and cutaneous microvascular endothelial cells (MECs). To determine which specific type of PBMLs bound to these r-IFN-gamma treated cells, we performed immunophenotyping on the adherent PBMLs. The adherent PBMLs were detached from the r-IFN-gamma treated keratinocytes and MECs by adding EDTA, and collected by cytocentrifugation, followed by immunocytochemical staining using a panel of monoclonal antibodies. Our results reveal that the relative adherent population of PBMLs was composed of approximately 60%-70% monocytes and 18%-24% Leu 2+ T lymphocytes (T-cytotoxic/suppressor) which preferentially bound to r-IFN-gamma treated keratinocytes and MECs. There was some lesser binding by Leu 3 + lymphocytes (T-helper/inducer); approximately 8%, and no binding of B lymphocytes. Since r-IFN-gamma also induced HLA-DR expression in keratinocytes and MECs, these in vitro data suggest that r-IFN-gamma may play an important role in the immunobiology of diverse skin diseases such as graft vs host disease, lichen planus, and other inflammatory dermatoses, because the keratinocytes express HLA-DR and the predominant T-cell subset in the epidermis is Leu 2 + (over the Leu 3 + T cell) in all of these conditions. These results represent a direct attempt to explain in situ immunophenotypic mononuclear leukocyte subset distribution patterns by using r-IFN-gamma and purified cultured cells such as keratinocytes and MECs. We propose that IFN-gamma, by both increasing the adherence of PBMLs, and promoting selective binding of monocytes and Leu 2 + T lymphocytes to both keratinocytes and MECs, may be important in regulating PBML localization and recirculation in the skin.
Preview · Article · Feb 1988 · Archives for Dermatological Research
[Show abstract][Hide abstract] ABSTRACT: To extend our previous observation in which the binding of resting allogeneic peripheral blood mononuclear leukocytes (PBML) to recombinant gamma-interferon (IFN-gamma)-treated keratinocytes was characterized, we examined the influence of phorbol ester activation of the PBML to both autologous and allogeneic IFN-gamma-treated keratinocytes. The activation of PBML by phorbol esters (5 to 100 ng/ml) for brief periods of time (5 min to 1 h) at 37 degrees C led to an increase in the relative percentage of adherence to IFN-gamma-treated keratinocytes from 15% for non-activated PBML to 30% for phorbol ester-treated PBML. A biologically inert phorbol ester derivative did not enhance the binding reaction. No significant binding of phorbol ester-activated PBML was observed to non-IFN-gamma-treated keratinocytes. Both reduction in temperature to 4 degrees C and preincubation of the phorbol ester-treated PBML with anti-LFA-1 monoclonal antibody, led to complete inhibition of this adherence reaction indicating a role for the LFA-1 molecule in phorbol ester-activated PBML/IFN-gamma-treated keratinocyte reactions. Immunophenotypic analysis of the adherent cell population of the phorbol ester-activated PBML to the IFN-gamma-treated keratinocytes revealed that the predominant adherent cell type was the CD8+ T-cell subset (44%) versus the CD4+ T-cell subset (33%) with 23% monocytes and no binding of B lymphocytes. These results suggest that phorbol ester-activated PBML binds twice greater than resting PBML to IFN-gamma-treated keratinocytes, and this increased adherence may further contribute to homing of activated lymphocytes to the epidermis and mononuclear cell trafficking in the skin of inflammatory dermatoses.
Preview · Article · Jun 1988 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: We investigated the in vivo effect of recombinant interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) treatment of mice on the development of the delayed-type hypersensitivity (DTH) reaction and lichenoid tissue reaction (LTR) following the local injection of cloned autoreactive T cells. Both the DTH reaction and the LTR were significantly enhanced by pretreatment with IFN-gamma, but not with TNF-alpha. Induction of class II MHC antigens on keratinocytes was not essential for the enhancement by IFN-gamma. Administration of anti-IFN-gamma antibody reduced the DTH reaction and LTR, although complete inhibition was not observed with our treatment regimen. The ability of IFN-gamma to increase the number of the cloned T cells invading the epidermis in vivo, is in keeping with our previous observation that IFN-gamma treatment of cultured keratinocytes markedly increased the adherence reaction between T cells and keratinocytes in vitro.
Preview · Article · Sep 1988 · British Journal of Dermatology