Mixed Epidermal Cell-lymphocyte Reaction Research Articles

Effect of Topical Sunscreens on the UV-Radiation–Induced Suppression of the Alloactivating Capacity in Human Skin In Vivo

Exposure of mice or humans to solar or artificial ultraviolet radiation (UV) has been shown to induce a number of changes in the immune system that may influence their susceptibility to skin tumors. The protective effect of sunscreens on these changes is not clear. Thirty-two patients with a variety of dermatoses routinely undergoing treatment with standard UVB (n = 19) or PUVA (n = 13) therapy were studied. One of the two tested sunscreens or its vehicle was applied to the right flexor forearm immediately prior to each total-body UV exposure. Epidermal sheets were obtained by the suction-blister method from the left flexor forearm before treatment and from both flexor forearms after 4 weeks of photo- or photochemotherapy and used as stimulator epidermal cells (EC) in the mixed epidermal cell-lymphocyte reaction (MECLR). After 4 weeks of either UVB or PUVA therapy the MECLR responses on EC from both arms were markedly decreased. Neither the tested sunscreens nor their vehicles prevented the UV-induced suppression of the alloactivating capacity. The failure of sunscreens to protect against the UV-induced suppression of the alloactivating capacity could be explained in two ways. First, the energy not absorbed by the sunscreen could be sufficient to induce suppression of the alloactivating capacity. An alternative explanation could be systemic immune suppression by UV. In order to discriminate between these possibilities only the right forearms of 10 healthy volunteers, treated with a sunscreen or its vehicle, were irradiated with UVB during 4 weeks. In this manner systemic immune suppression by UVB could be excluded. This experiment resulted in a similar suppression of the MECLR responses, as induced by total body UVB irradiation, without any protection by the sunscreen. Apparently, the UV dose not absorbed by the sunscreen was capable to induce suppression of the alloactivating capacity. Our results indicate that people protected from sunburn by sunscreens may be exposed to UV for a long period of time, and thereby subject themselves to its immunosuppressive action.

Cyclosporin A Inhibits the Antigen-Presenting Functions of Freshly Isolated Human Langerhans Cells In Vitro

Cyclosporin A (CsA) is a strong inhibitor of skin allograft rejection. It has been also reported to act, not only on helper T cells, but also on the antigen-presenting functions of mouse epidermal cells (EC) enriched in Langerhans cells (LC). We tested the effects of CsA on the human allogeneic mixed epidermal cell-lymphocyte reaction (MECLR) using whole EC and freshly isolated LC as stimulator cells. Results were as follows. 1) CsA inhibited the lymphocyte proliferative response in a dose-dependent fashion, by about 80% for a CsA concentration of 10(-7) M. To evaluate the effects of the drug on the two cell populations involved in MECLR, stimulator EC and responder PBMC were separately pulsed for 2 h with CsA, washed, and combined to form MECLR. Inhibition by CsA of the alloantigen-dependent lymphocyte proliferation appeared to be multifactorial, because CsA-pulsed EC and CsA-pulsed effector PBMC led to identical reductions of 40% each in proliferation. 2) The nature of EC sensitivity to CsA during MECLR was then analyzed after freshly separating highly purified CD1-positive LC (greater than 95%) and LC-depleted EC (mainly keratinocytes), using an immunomagnetic particle technique. When responder PBMC were cultured with CsA-pulsed LC, a highly significant reduction of lymphocyte proliferation was observed, indicating that CsA has direct effects on LC. 3) Some of the possible mechanisms by which CsA might act on LC were studied. Substantial IL-1 activity and PGE2 amounts were induced during MECLR by LC and keratinocytes, but CsA did not act via these factors. Neither did it significantly modify HLA-DR, DQ, or DP antigen expressions on EC. In conclusion, CsA directly inhibits antigen presentation by human LC. This inhibition may partly explain the beneficial effects of CsA on skin allografts and certain cutaneous immune reactions.

Granulocyte Macrophage-Colony-Stimulating Factor (GM-CSF) Decreases CD1a Expression by Human Langerhans Cells and Increases Prolifereation in the Mixed Epidermal Cell-Lymphocyte Reaction (MELR)

Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro. To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines. The percentage of cells expressing the surface protein CD1a was determined by flow cytometry. This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value. By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures. Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a. Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation. To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured. Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells. GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines. We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.

Synthetic Retinoids Inhibit the Antigen Presenting Properties of Epidermal Cells in Vitro

The clinical efficacy of retinoids in benign and malignant skin diseases involving immune mechanisms suggests that they affect the immunologic functions of the epidermis. However, these effects have yet to be demonstrated. The action of vitamin A (retinol) and the synthetic retinoids, isotretinoin, etretinate, acitretin, and arotinoid-free acid have been studied on the lymphocyte proliferation induced by phytohemagglutinin (PHA), by the mixed lymphocyte reaction (MLR), and the mixed epidermal cell-lymphocyte reaction (MECLR). The results for PHA-induced proliferations were highly variable for all the retinoids. However, in MECLR, the synthetic retinoids consistently reduced the proliferation by 20%-30%. This occurred at therapeutic drug concentrations of about 10(-7)M. In MLR, a minor decrease of 10%-15% was only found for higher concentrations (10(-5)M). Retinol induced no effect in either reaction. Further analysis of acitretin on MECLR showed that it reduced lymphocyte proliferation in a dose-dependent fashion. This reduction was combined with a decrease in cytotoxic T-lymphocyte induction (CTL). Addition of 10(-6)M acitretin at various times also revealed that its presence at cell culture initiation was necessary to inhibit proliferation significantly. Furthermore, cell treatments prior to MECLR showed that exposure of epidermal cells to acitretin was essential to produce this inhibition, suggesting that it acts directly on epidermal cells. Consequently, it is suggested that the specific inhibitory effect of synthetic retinoids on lymphocyte activation in MECLR may partly account for their therapeutic action on the skin.

The mixed epidermal cell lymphocyte‐reaction is the most predictive factor of acute graft‐versus‐host disease in bone marrow graft recipients

Risk factors for acute graft-versus-host disease (GvHD) remain controversial. We performed uni- and multivariate statistical analyses on a series of 37 patients receiving a non-depleted allogeneic bone marrow transplant from an HLA-identical sibling donor for a haematological malignancy, in order to identify risk factors for GvHD. Three factors were associated with development of moderate to severe GvHD: a positive mixed epidermal cell-lymphocyte reaction (MECLR) between donor and recipient, previous pregnancies in female donors and chronic myeloid leukaemia diagnosis. The MECLR was the most important predictive factor, selected in first rank by the stepwise linear discriminant analysis. Combining these three prognostic factors in the jackknifed procedure, we could correctly classify 33/37 patients in two groups: grade O-I versus grade II-IV acute GvHD. These results should apply to donor selection and to predict donor/recipient pairs at high risk of GvHD who might benefit of bone marrow T-cell depletion and those at low risk for whom depletion could be avoided.

Lympho‐epidermal Interactions in Patients Receiving Human Langerhans cell Free Cultured Epidermal Allografts as a Skin Substitute

AbstractHuman Langerhans cell free epithelia can be cultured in vitro, and then can be used as epidermal allografts (EAG) without evidence of rejection. We studied the cellular basis of this phenomenon with mixed epidermal cell lymphocyte reactions (MELR). The capacity of donor‐derived epidermal cells to stimulate allogeneic control or recipient cells was abolished when stimulatory cells were Langerhans cell‐free cultured keratinocytes and respondors were obtained prior to grafting. Donor‐type cultured keratinocytes were able to induce a low response by host‐derived cells in assays conducted 2, 4 and 6 weeks after grafting, but not thereafter. They were unable to stimulate allogeneic cells unrelated to the recipient. The ability of crude epidermal suspensions with 2–4% Langerhans cells to stimulate host‐derived cells did not increase with time after grafting. No secondary type MELR could be evidenced, suggesting that grafting of EAG did not induce an in vivo immunization to class I antigens expressed by cultured epidermal cells. Lastly, host‐derived Langerhans cells were not able to restore the allostimulatory ability of Langerhans cell free epidermal cells from EAG donors when tested against host‐derived cells. This suggests that host‐derived Langerhans cells which colonize the grafts during the first few weeks following grafting cannot act in the presentation of foreign keratinocyte‐bound antigens, which may account for the absence of rejection noted.

Gamma interferon modulates epidermal cell proliferation and mixed epidermal cell-lymphocyte reaction.

Gamma interferon (gamma-IFN), a lymphokine produced by activated T lymphocytes, has a variety of effects on target cell. It induces class II antigens of the major histocompatibility complex not only in immunocompetent cells but also in non-immunocompetent cells. gamma-IFN also can exert, in addition to anti-viral activity, a series of anticellular effects on a variety of cell types. The effects of gamma-IFN on the proliferation of cultured epidermal cell (EC) and induction of HLA-DR antigen expression by EC (HLA-DR+KC) were studied. Furthermore, the immunologic role of HLA-DR+KC in the mixed epidermal cell-lymphocyte reaction (MECLR) was studied. The antiproliferative effect of gamma-IFN on the cultured EC was seen 3 days after treatment of gamma-IFN and the effect was dose-dependent. Number of HLA-DR+KC was increased dose-dependently with treatment of gamma-IFN. In MECLR, HLA-DR+KC had been found to exert stimulatory role on allogenic lymphocytes. However, there was no significant role of HLA-DR+KC on autologous lymphocytes.

Stimulation of T cells by autologous mononuclear leukocytes and epidermal cells in psoriasis

Based on reports suggesting aberrant cell-mediated immunity and altered infiltration of immunocompetent cells into the skin in psoriasis, we studied the stimulation of T cells by autologous non-T mononuclear leukocytes (autologous mixed lymphocyte reaction, AMLR) and by epidermal cells isolated from lesional and clinically uninvolved skin in psoriasis (autologous mixed epidermal cell lymphocyte reaction, AMECLR). Age- and sex-matched individuals served as controls. We found that the AMLR in psoriasis (n = 11) was similar to that in healthy controls (n = 16); furthermore, cell proliferation was alike in the presence of either 5% AB-serum or autologous serum. By contrast, while the AMECLR in healthy controls (n = 9) resembled that in psoriatics employing epidermal cells from univolved skin, epidermal cells from lesional sites (n = 10) induced a significantly higher proliferation of autologous T cells in the AMECLR (P less than 0.01). We conclude that the in vitro stimulation of T cells by non-T mononuclear leukocytes is normal in psoriasis and is not regulated by autologous serum. Lesional psoriatic epidermal cells, however, are more active in stimulating autologous T cell proliferation than cells from univolved psoriatic or normal epidermis.

Mixed Epidermal Cell-Lymphocyte Reaction in Prediction of Acute Graft-Versus-Host Disease in Bone Marrow Recipients<xref ref-type="fn" rid="FN1">1</xref>

Peripheral blood lymphocytes (PBL) and epidermal cells (EC) of 44 patients to be grafted with bone marrow from an HLA-identical sibling have been used as stimulator cells in primary cultures with effector PBL of one or several potential donors. Proliferative responses against PBL did not differ from those obtained with effector cells cultured in medium alone, whereas EC induced clearly positive proliferation in 21/53 (40%) of the pairs tested. Evaluation of 30 patients followed for more than 3 months after the graft shows that a high level of response in the mixed epidermal cell lymphocyte reaction is directly correlated with the incidence of acute graft-versus-host disease.

A possible predictive test for graft-versus-host disease in bone marrow graft recipients: the mixed-epidermal cell-lymphocyte reaction.

The possible ability of the mixed-epidermal-cell-lymphocyte reaction (MECLR) to detect alloreactivities in HLA-identical MLR negative siblings has been investigated before grafting in 30 donor-recipients pairs and their families. Results indicate that recipients' epidermal cells (EC) can induce proliferative responses of HLA-identical MLR-negative donors' lymphocytes in 55% of the pairs tested. Moreover, further evaluation of 21 patients shows that the positivity of the MECLR before the graft is correlated with later appearance of acute and chronic GVHD, and especially with the severity of cutaneous injury. EC have been shown to be more efficient antigen presenting cells than peripheral blood lymphocytes for in vitro primary proliferative responses, so these reactions could be directed against some minor histocompatibility antigens, thus leading to possible improvement in selecting bone marrow graft donors and to the detection of donor-recipient pairs with a high risk of GVHD.

The mixed epidermal cell-lymphocyte reaction. I. Human epidermal cells elicit a greater allogeneic lymphocyte response than do autologous peripheral blood lymphoid cells.

In contrast to studies by other investigators, it was observed that epidermal cells isolated from flexor forearm skin of normal adult human volunteers are consistently superior to autologous peripheral blood mononuclear cells in triggering allogeneic lymphocytes to undergo blastogeneic transformation. Stimulator cell dilution studies revealed an eightfold greater allogeneic lymphocyte-stimulating potency for epidermal cells when compared to autologous peripheral blood mononuclear cells. This difference was shown not to have resulted from enzyme exposure during cell isolation procedures or suppressor cells present in the stimulator peripheral blood mononuclear cell populations. Our technique for isolating epidermal cells is straightforward and is harmless enough to make it reasonable to consider using the mixed epidermal cell lymphocyte reaction in matching human allograft donors and recipients.

Differential Distribution of Langerhans Cells in Organ Culture of Human Skin

Epidermal Langerhans cells (LCs) function as the antigen-presenting cells in such cutaneous cell-mediated immune responses as contact hypersensitivity and in the mixed epidermal cell-lymphocyte reaction. They have also been implicated in the immune response in skin allograft rejection. Since organ culture of thyroid and pancreas has been shown to prolong allograft survival, presumably through the loss of antigen-presenting cells, we examined the effect of skin explant culture on LC survival. Human skin explants were placed in organ culture and examined serially as whole mounts of epidermis for the presence of LCs as judged by ATPase activity, and OKT-6 and HLA-DR antigens. Although we observed morphologic changes and an absolute reduction in the number of positively stained cells, culture for up to 28 days failed to deplete explants of these cells. Langerhans cells were also sought in the epidermal outgrowths that develop peripheral to the original explants. They were never seen in the area beyond 0.3 mm from the explant edge. Organ culture of skin thus provides a means to explore the contribution of LCs to skin allograft rejection by comparing the immunogenicity of epidermal portions of the explant with the epidermal outgrowth.