Rat spleen dendritic cells express natural killer cell receptor protein 1 (NKR-P1) and have cytotoxic activity to select targets via a Ca2+-dependent mechanism.

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95-98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand-Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.

Indirect CD4+ TH1 response, antidonor antibodies and diffuse C4d graft deposits in long-term recipients conditioned by donor antigens priming.

Priming of recipients by DST induces long-term survival of mismatched allografts in adult rats. Despite these recipients developing inducible T regulatory cells able to transfer long-term graft survival to a secondary host, a state of chronic rejection is also observed. We revisited the molecular donor MHC targets of the cellular response in acute rejection and analyzed the cellular and humoral responses in recipients with long-term graft survival following transplantation. We found three immunodominant peptides, all derived from LEW.1W RT1.D(u) molecules to be involved in acute rejection of grafts from unmodified LEW.1A recipients. Although the direct pathway of allorecognition was reduced in DST-treated recipients, the early CD4+ indirect pathway response to dominant peptides was almost unimpaired. We also detected early and sustained antidonor class I and II antibody subtypes with diffuse C4d deposits on graft vessels. Finally, long-term accepted grafts displayed leukocyte infiltration, endarteritis and fibrosis, which evolved toward vascular narrowing at day 100. Altogether, these data suggest that the chronic graft lesions developed in long-term graft recipients are the result of progressive humoral injury associated with a persisting indirect T helper response. These features may represent a useful model for understanding and manipulating chronic active antibody-mediated rejection in human.

An immunomodulatory role for follistatin-like 1 in heart allograft transplantation.

Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8(+) T cells and TGFbeta are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8(+) T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFNgamma. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance.

IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis.

Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

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.

Production of paf-acether by human epidermal cells.

The production of the inflammatory mediator paf-acether (paf) from human epidermal cells was investigated in vitro. Human epidermal cells, freshly isolated from normal skin or in culture, were incubated in Tyrode's buffer containing 0.25% lipid-free bovine serum albumin in the presence of 2 microM calcium ionophore A23187, at 37 degrees C, for 1 to 60 min. Paf production slightly began at the first min of stimulation, was significant after 10 min, reached a maximum at 20 min (251 +/- 25 pg/l X 10(6) cells, mean +/- 1 SD), and decreased thereafter. About 50% of the paf amount produced by epidermal cells was recovered in supernatants. Addition of the non-acetylated paf precursor 1-O-octadecyl-sn-glycero-3-phosphocholine, i.e., lyso-paf, at 0.1 microM to epidermal cells during A23187-stimulation did not alter this production. In contrast, addition of acetyl-coenzyme A at 0.1 mM enhanced paf production by 5 times. The material produced by epidermal cells was identical to synthetic paf because: 1) the aggregation of aspirin-treated and ADP-insensitive washed rabbit platelets it induced was inhibited by BN 52021, an antagonist of the paf putative receptor; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse-phase (RP) high-pressure liquid chromatography (HPLC) elution. The paf precursors, i.e., lyso-paf and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, were also detected in epidermal cells, stimulated with A23187 or not. As determined by RP-HPLC analysis and confirmed by gas chromatography analysis, these precursors and the paf produced by epidermal cells exhibited more than 90% of a hexadecyl chain at the sn-1 position of the molecule. The present results demonstrate the synthesis and release of paf by normal human epidermal cells. Paf production within the epidermis might account for the development of cutaneous inflammation and the pathogenesis of many skin disorders.

T-cell receptor-gamma/delta bearing lymphocytes in normal and inflammatory human skin.

Murine dendritic epidermal T cells (DETC) were recently reported to express T-cell receptor (TCR)-gamma/delta chains. In a search for the human equivalent of these cells, we tested normal and lesional skin with MoAb which react with the TCR-gamma/delta heterodimer. We performed indirect immunofluorescence (IF) on epidermal sheets, and alkaline-phosphatase-anti-alkaline-phosphatase complex (APAAP) on epidermal cell smears. Frozen skin sections from normal skin and various cutaneous lymphocyte infiltrates were also studied. A few CD3+ T lymphocytes were consistently found in normal epidermis. Most of these cells appeared to be TCR-alpha/beta +, and some CD4+ or CD8+. On epidermal sheets and cell smears, only a very small TCR-gamma/delta + cell population was visualized (less than 0.1% of the total). On normal skin sections, we observed 0 to 3 gamma/delta + cells per section. When present, they were often located in the epidermal basal layer, and were round or dendritic. Double immunolabeling revealed that gamma/delta + cells differed from CD1+ Langerhans cells, and that they had a similar phenotypic pattern as gamma/delta + peripheral lymphocytes (PBL): CD2+, CD3+, CD4-, and CD8-. Immunostaining from various inflammatory skin lesions showed that the dermal infiltrates included CD3+ T lymphocytes but virtually no gamma/delta + cells. Only a few gamma/delta + cells were found in some end-evolutive infiltrates. Taken together, these results strongly suggest that normal human epidermis occasionally harbors TCR-gamma/delta complex bearing lymphocytes, which constitute a small fraction of the CD3+ cutaneous T lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Impaired antigen presentation in toxic epidermal necrolysis.

BACKGROUND AND DESIGN--The pathophysiology of toxic epidermal necrolysis (TEN) remains largely unknown. Toxic epidermal necrolysis is considered a hypersensitivity reaction to drugs, but direct evidence of an immunologic mechanism is still lacking. Lymphopenia and a decrease in the numbers of circulating CD3- and CD4-positive lymphocytes have been reported in acute phase, but, to our knowledge, no study of cellular immune functions of patients with TEN has been reported so far. Herein, we investigated several T-cell functions in a series of 11 patients with TEN. Peripheral blood mononuclear cells (PBMC) obtained in the acute phase were tested together with PBMC obtained after the patient's recovery and compared with those of age- and sex-matched healthy control subjects. RESULTS--Phytohemagglutinin-induced proliferations and lymphocyte responses in allogeneic mixed lymphocyte reactions were not impaired in the acute phase compared with those after recovery in the same patients and with those in control subjects. In contrast, natural killer cytotoxicity and allogeneic cytotoxic responses were significantly decreased in early TEN. The most striking feature was the significantly impaired ability of acute-phase lymphoid cells to activate allogeneic T cells. Patient PBMC in acute-phase TEN did not inhibit the proliferation induced by patient PBMC after recovery, suggesting that their defect was not related to the presence of radioresistant suppressor cells. The phenotypic expression of HLA-DR, -DQ, and -DP antigens on circulating peripheral blood lymphocytes was then assessed by immunoalkaline phosphatase staining and flow cytometry. Results showed decreased percentages of HLA-DR-positive mononuclear cells and a decreased density of HLA-DR antigens, mainly on monocytes, in acute-phase TEN. CONCLUSIONS--These results demonstrate that peripheral blood lymphocytes of patients with TEN have an impaired ability to activate allogeneic T cells. This defective antigen presentation is not secondary to the presence of suppressor lymphocytes, but it is probably related to a decreased expression of HLA-DR antigens on circulating mononuclear cells in acute-phase TEN.

[Mixed lympho-epidermal cell cultures: value in bone marrow grafts]. / Cultures mixtes lympho-épidermiques: intérêt dans les greffes de moelle.

Graft-versus-host disease (GVHD) is the major complication of allogeneic HLA-identical bone marrow transplantation. GVHD is induced by the activation of mature T cells in the graft which react against minor antigens of the recipient. Mixed epidermal cell-lymphocyte cultures (MELC), which constitute an in vitro model of epidermal cell-lymphocyte interactions, make it possible to study the presentation of antigens to the lymphoid cells by epidermal Langerhans cells. We performed MELC in 66 patients who had received an HLA-identical bone marrow transplant for malignant blood disease. The bone marrow received by 29 recipients had been depleted of mature T cells, whereas 37 recipients had received a non-depleted bone marrow. A complete, uni- and multivariate statistical analysis was carried out on recipients of non-depleted bone marrow to evaluate the risk factors for acute and chronic GVHD. This study showed that MELC between donor and recipient was the most predictive parameter for the occurrence of GVHD. Other factors were a history of previous pregnancies in female donors and a diagnosis of chronic myelogenous leukaemia. These results may be of value in the selection of donors and for a better determination of the need for bone marrow depletion.

Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid.

Interleukin-22 (IL-22) is mainly produced at barrier surfaces by T cells and innate lymphoid cells and is crucial to maintain epithelial integrity. However, dysregulated IL-22 action leads to deleterious inflammation and is involved in diseases such as psoriasis, intestinal inflammation, and cancer. IL-22 binding protein (IL-22BP) is a soluble inhibitory IL-22 receptor and may represent a crucial regulator of IL-22. We show both in rats and mice that, in the steady state, the main source of IL-22BP is constituted by a subset of conventional dendritic cells (DCs) in lymphoid and non-lymphoid tissues. In mouse intestine, IL-22BP was specifically expressed in lamina propria CD103(+)CD11b(+) DC. In humans, IL-22BP was expressed in immature monocyte-derived DC and strongly induced by retinoic acid but dramatically reduced upon maturation. Our data suggest that a subset of immature DCs may actively participate in the regulation of IL-22 activity in the gut by producing high levels of IL-22BP.

Role of IFNgamma in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

Cantharide acantholysis: endogenous protease activation leading to desmosomal plaque dissolution.

Using a method which allowed us to study the morphological consequences of the expression and the inhibition of proteases in living tissues, we demonstrated that the primary detectable cellular event in cantharide acantholysis is the dissolution of the dense plaque, leading to the detachment of tonofilaments from desmosomes. This process is inhibited by neutral serine protease inhibitors. This suggests that the desmosome-tonofilament complex, more precisely the desmosomal dense plaque, is the primary target of activated proteases during cantharide acantholysis, and can be disrupted by a specific epidermal protease-anti protease system. Cantharide acantholysis may be useful model for studying desmosomal turnover.

Reconstructed human epidermis: absence of Langerhans cells and failure to stimulate allogeneic lymphocytes in vitro.

Whole human skin can be reconstructed in vitro, using dermal equivalents made of fibroblasts in a collagen matrix. We recently described a new method of epidermalization of dermal equivalents, based on the insertion of punch biopsies and the migration of epidermal cells (EC) on the reconstructed dermis. In the present study, we show that no MHC class II or T6 positive Langerhans cells (LC) can be detected in this new epidermis. Functional studies with EC of this reconstructed epidermis show that these EC completely fail to induce proliferation of allogeneic lymphocytes in mixed epidermal cell lymphocyte reactions and to raise an allogeneic T cell response. In contrast, fresh EC from the same donors induce a strong proliferative and cytotoxic response of the same effector cells. Moreover, the addition of fresh LC-containing EC autologous to effector lymphocytes does not restore an allogeneic proliferative and cytotoxic response directed against class I different EC of the new epidermis. Such a non-immunogenic whole skin model composed of two compartments, dermis and epidermis, completely devoid of class II-bearing antigen presenting cells, is thus a very promising technique for allogeneic skin grafting in the treatment of burns.

Critical requirement for graft passenger leukocytes in allograft tolerance induced by donor blood transfusion.

Tolerance to a vascularized allograft can be induced in adult animals by pregraft donor-specific blood transfusion (DST). Mechanisms underlying this effect appear to depend on unresponsiveness of alloreactive T-helper cells. In this study, we examined the roles of DST and cellular components of the allograft that are important in inducing T-cell unresponsiveness in a rat model. DST alone did not tolerize alloreactive recipient T-helper cells, but the combination of DST and heart allograft induced profound inhibition of the antidonor proliferative response in spleen but not in lymph node cells. When heart allografts were depleted of passenger leukocytes by pretreating the donor with cyclophosphamide or by parking the graft for 2 months in a tolerant recipient, tolerance induction in DST-treated recipients was abrogated. Tolerance could then be restored in a majority of DST-treated recipients of passenger leukocytes depleted grafts by injecting them at the time of grafting with donor, but not third-party, dendritic cells. This indicates that graft passenger leukocytes, most likely dendritic cells, are required for DST-induced allograft tolerance.

Human epidermal cells are more potent than peripheral blood mononuclear cells for the detection of weak allogeneic or virus-specific primary responses in vitro.

Human epidermal cells (EC) and peripheral blood mononuclear cells (PBMC) have been used as antigen-presenting cells in allogeneic reactions or in self-restricted antiviral responses. Comparison of results from both cell types indicates that: (1) EC were better stimulators of primary proliferative responses in all the antigenic systems tested. (2) In secondary reactions, EC and PBMC functioned similarly for allogeneic responses, while a weak but significant difference could be observed in both (HSV1 or influenza A) virus-specific reactions. (3) By comparing pairs of HLA-identical mixed lymphocyte reaction (MLR)-negative siblings, positive responses were observed in several different families when lymphocytes of potential bone marrow donors were stimulated by EC of the recipient. This suggests that EC might be useful in detecting relatively weak proliferative responses in a number of antigenic systems, but especially in primary reactions against viral or putative minor histocompatibility antigens. (4) Despite this stronger antigen-presenting capacity in proliferative responses, EC induced lower levels of cytotoxic T lymphocyte (CTL) reactions than PBMC, not only in allogeneic responses but also in virus-specific self-restricted reactions.

A new method for studying epidermalization in vitro.

A new method for studying epidermalization in vitro is described. It consists of inserting a punch biopsy that serves as a source of epidermis into dermal equivalent freshly made up, with fibroblasts mixed in a collagen matrix. Fibroblasts cling to collagen fibrils and contract the matrix, leading in 3 days to a resistant dermal equivalent holding the punch biopsy firmly in place. At day 5, a culture medium favouring epidermal growth was used and a fringe of a new epidermis appeared around the punch, the area of which grew linearly with time. This new epidermis showed a pattern of differentiation similar to epidermis in vivo, with cuboidal basal cells, keratohyalin granules, membrane coating granules and the expression of the 65-67 kd keratin subset. The method seems to combine the advantages of the explant technique and of classical keratinocyte cultures, providing the researcher with a large quantity of differentiated epidermis, the pharmacologist with simple and quantitative system in which to study modifications of growth and differentiation of epidermis, and the plastic surgeon with a possible material for skin grafting.

Recombinant IFN-gamma abrogates allograft tolerance induced by donor-specific blood transfusion by restoring alloantibody production.

Donor-specific tolerance to heart allograft was induced in adult Lewis rats by pregraft donor-specific blood transfusion (DST). We previously showed that this tolerant state is characterized by a dramatic inhibition of T cell and macrophage activation. In addition, tolerant animals could not mount an efficient anti-donor humoral response whereas transfer of sera from rejecting animals triggered rejection in tolerant animals. This tolerance can be abrogated by daily post-graft administration of recombinant IFN-gamma (rIFN-gamma). To elucidate the mechanisms of action of rIFN-gamma, T cell, macrophage and B cell functions were assessed in allograft recipients. IFN-gamma did not restore the expression of Th1-related cytokine mRNA or the activated macrophage product inducible nitric oxide synthase in allografts. Importantly, rIFN-gamma treatment promptly restored the anti-donor humoral response in DST-treated recipients. We conclude that rIFN-gamma treatment in DST-treated allograft recipients cannot reverse the unresponsive state of Th1 cells and macrophages infiltrating the graft, but can provide B cell help for IgG alloantibody production which is lacking in these animals.

Involvement of macrophages in the pathology of toxic epidermal necrolysis.

In toxic epidermal necrolysis (TEN), as in the 'epidermal type' of erythema multiforme, the necrotic epidermis is infiltrated with mononuclear cells. We studied the epidermal infiltrate in seven cases of TEN. About half the cells obtained from pieces of cleaved epidermis dissociated by trypsin were non-epithelial. On cytologic analysis, 80% of these foreign cells exhibited markers of macrophages, 15% were granulocytes and only 5% were lymphocytes (almost exclusively OKT8 T lymphocytes). Semi-thin sections of early prenecrotic lesions showed exocytosis of mononuclear cells within the epidermis with features of satellite cell necrosis and formation of colloid bodies. Almost all these mononuclear cells were macrophages as evidenced by endogenous peroxidase-positive granules. These findings suggest that some kind of macrophage-mediated cytotoxicity may play a role in the necrosis of epidermal cells during TEN.