bromochloroacetic-acid and Graft-vs-Host-Disease

Graft-versus-host disease (GVHD) occurs when lymphoid cells from an immunocompetent donor are introduced into a histoincompatible recipient that is incapable of rejecting them. GVHD is seen most commonly as a complication of therapeutic bone marrow transplantation, and the skin is a primary target organ. As GVHD mimics several important skin diseases both clinically and histologically, it provides a useful model for studying the immunopathologic features of epidermal cell-lymphocyte interactions.

Topics: Animals; Epidermis; Graft vs Host Disease; HLA-D Antigens; Humans; Keratins; Langerhans Cells; Skin Diseases; T-Lymphocytes

Killer cell immunoglobulin-like receptors (KIRs) recognize different groups of Human Leukocyte Antigen (HLA) class I alleles and are expressed by natural killer (NK) cells and some T lymphocytes. NK cell cytotoxicity is triggered by failure to recognize the appropriate HLA class I ligand on target cells. Recently, it has been shown that HLA class I ligand incompatibility in the graft-versus-host (GvH) direction is associated with a better outcome in haploidentical hematopoietic stem cell transplantation (HSCT). Since KIR genotypes are very diverse in the population, we explored whether or not the donor KIR genotype could affect the graft-versus-leukemia (GvL) effect in the related HLA-identical HSCT setting. We determined the KIR and HLA genotypes of 65 HLA-identical patient-donor siblings. We found that the presence of two activating KIRs, 2DS1 and 2DS2, in the donor was significantly associated with a decreased leukemic relapse rate (P=0.03; OR=0.18; 95% CI: 0.037-0.88). Moreover, the probability of relapse at 5 years was significantly lower for patients who received a graft from a donor with the 2DS1(+)2DS2(+) genotype than for those who received a transplant from other donors (17 vs 63%, respectively; P=0.018). In conclusion, this study suggests that a joint effect of these two selected activating KIRs in the donor might confer some protection against leukemic relapse.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Genotype; Graft vs Host Disease; Graft vs Leukemia Effect; Hematopoietic Stem Cell Transplantation; Histocompatibility; Histocompatibility Testing; Humans; Keratin-2; Keratins; Killer Cells, Natural; Leukemia; Leukocyte Transfusion; Male; Middle Aged; Receptors, Immunologic; Receptors, KIR; Secondary Prevention; Survival Analysis

To understand the mechanisms involved in immunological tolerance to skin-associated antigens, we have developed transgenic (Tg) mice that express a model self-antigen, membrane-bound chicken ovalbumin (OVA), under the control of a keratin 14 (K14) promoter. K14-mOVA Tg mice express OVA mRNA in the epidermis, and appear normal. K14-mOVA Tg mice failed to mount T cell and delayed type hypersensitivity reactions to OVA, suggesting that the Tg mice were tolerant to OVA. Skin dendritic cells, including Langerhans cells, may contribute to the tolerance induction because migratory skin DC derived from K14-mOVA efficiently activated CD8(+) T cells from OVA-specific T-cell receptor (Va2/Vb5) Tg (OT-I) mice. OT-I cells expanded and accumulated in skin-draining lymph nodes after intravenous injected into K14-mOVA mice and exhibited activation markers. Graft-versus-host disease-like skin lesions appeared in K14-mOVA mice by day 7 after injection of OT-I cells. These studies demonstrate that K14-mOVA Tg mice are susceptible to an autoimmunelike skin disease induced by passively transferred naïve CD8(+) OVA T-cell receptor Tg T cells, and serve as a good model for understanding self-tolerance and for the investigation of the pathogenesis, treatment and potential prevention of cell-mediated autoimmune reactions in skin.

Topics: Adoptive Transfer; Animals; Autoantigens; CD8-Positive T-Lymphocytes; Cell Movement; Gene Expression; Graft vs Host Disease; Hypersensitivity, Delayed; Keratin-14; Keratins; Mice; Mice, Transgenic; Ovalbumin; Promoter Regions, Genetic; Skin Diseases; Transgenes

The identity of cells within squamous epithelia that represent primary targets in acute graft-versus-host disease (GVHD) has been an enigma. Murine effector T cells implicated in the alloresponse by Vbeta complementarity-determining region-3 spectratype analysis were detected with a Vbeta-specific monoclonal antibody within discrete microdomains of tongue (lingual) squamous epithelium. These microdomains, termed rete-like prominences (RLPs), are similar to the rete ridges of human skin. Cells forming the basal layer of RLPs and of human skin rete ridges were shown to express a distinctive pattern of keratin expression defined by antibodies to cytokeratin 15 (K15). In experimental murine GVHD elicited across minor histocompatibility antigen barriers (miHA), early lesions involved selective apoptosis and loss of K15(+) staining within lingual RLPs. An in vitro organ culture model designed to investigate target cell injury by short-term exposure to tumor necrosis factor-alpha and interleukin-1beta, mediators relevant to GVHD, showed a similar pattern of apoptosis and loss of K15(+) reactivity within RLPs. In aggregate, these findings establish a novel cytoskeletal marker for target epithelial subpopulations that should facilitate evaluation of mechanisms of host cell injury in GVHD. These data may also enable the development of therapeutic approaches to abrogate disease at the level of target cell blockade.

Topics: Animals; Apoptosis; Bone Marrow Transplantation; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Movement; Cell Survival; Complementarity Determining Regions; Cytokines; Disease Models, Animal; Epithelial Cells; Flow Cytometry; Gene Expression; Graft vs Host Disease; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-1; Keratin-15; Keratins; Lingual Frenum; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Microscopy, Electron; Microscopy, Fluorescence; Peptide Fragments; Receptors, Antigen, T-Cell, alpha-beta; Skin; T-Lymphocytes; Tumor Necrosis Factor-alpha

When graft-versus-host (GVH) disease affects the liver, it is characteristically the bile ducts which are involved, infiltrated by lymphocytes. To characterize this process further, and to determine whether there were any antigenic changes in the bile ducts, we stained 9 liver biopsies involved by GVH disease, 10 non-GVH biopsies that had a prominent portal lymphocytic component, and 8 biopsies taken incidentally at surgery for noninflammatory liver disease with epithelial membrane antigen, AE-3, AE-1, a keratin cocktail, keratin 19, CD45RO (UCHL-1), CD43 (Leu-22), CD20 (L26), vimentin, and LN-3. The infiltrating lymphocytes were T cells (CD45RO+, CD43+, CD20-) which variably expressed LN-3. The bile ducts were positive for the keratin cocktail, AE-1, AE-3, and keratin-19, but only occasionally positive for EMA and LN-3. There was no significant difference in the staining patterns of either the bile duct cells or lymphocytes between the three groups. With the antibodies that we used, there does not appear to be a significant difference in the antigenic phenotype of the bile ducts in GVH as compared to normal or reactive livers.

Topics: Antigens, CD; Bile Ducts; Biopsy, Needle; Graft vs Host Disease; Humans; Immunohistochemistry; Immunophenotyping; Keratins; Liver Transplantation; Lymphocytes; Retrospective Studies

Nine marrow allograft recipients who developed GVHD of the gastrointestinal tract accompanied by the passage of ropey necrotic material per rectum were studied. The material, resembling necrotic intestinal mucosa, was evaluated for the presence of epithelial cells using monoclonal antibodies for keratin and macrophages. Two keratins (AE1/AE3 and 34 beta E12) were detected within cells in all cases while macrophages were found in all but one case. In three cases, some cells were positive for both antibodies suggesting the presence of phagocytosed keratin in macrophage cytoplasm. Search for organisms with Gram and methenamine silver stains showed bacteria in six cases and fungus in one. Some cases of severe GVHD will be associated with the passage of ropey tan material grossly resembling sloughed mucosal tissue, but microscopically consisting largely of fibrin clots. The presence of free intestinal epithelial cells in this material is confirmed by the present study.

Topics: Bone Marrow Transplantation; Epithelium; Graft vs Host Disease; Humans; Immunohistochemistry; Intestinal Mucosa; Intestines; Keratins; Necrosis

Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.

Topics: Animals; Base Sequence; Cachexia; Graft vs Host Disease; Keratins; Mice; Mice, Transgenic; Microscopy, Electron; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Promoter Regions, Genetic; Skin; Tumor Necrosis Factor-alpha; Up-Regulation

Sweat gland abnormalities occur much more frequently than hitherto described in cutaneous graft versus host disease (GVHD). Two patterns of abnormalities were identified in 80 per cent of cases of acute GVHD: a cytopathic pattern consisting of a combination of basal vacuolopathy with or without lymphocytic infiltration and basal cell degeneration, and a proliferative pattern consisting of basal cell hyperplasia. In chronic GVHD, complete sweat gland destruction with fibrosis was commonly observed. Squamous metaplasia and dilation of the sweat glands were less frequently identified. Ki67 immunostaining confirmed proliferative activity in the basal cells of the distal duct. HLA-DR antigens were expressed on the basal cells of the duct and secretory glands in acute GVHD but not in normal skin. Langerhans cells were absent in both normal and abnormal sweat glands. The role of HLA-DR or Langerhans cells in the initiation of GVHD is questioned in the light of the new data and the primary involvement of proliferating cells is confirmed.

Topics: Acute Disease; Adolescent; Adult; Aged; Cell Survival; Child; Child, Preschool; Chronic Disease; Female; Graft vs Host Disease; Humans; Hyperplasia; Keratins; Male; Middle Aged; Skin Diseases; Sweat Glands

We have used monoclonal antibodies to study the expression of calgranulins by keratinocytes in inflammatory dermatoses. Calgranulins are intracellular calcium binding proteins which have inflammatory cytokine activity and are composed of at least two different chains, calgranulin A and B. Antibody CF 145 and CF 557 identify calgranulin A and B, respectively. MAC 387 recognizes a molecule probably containing both calgranulins. Keratinocytes in normal skin did not contain these molecules. The keratinocytes in 52 cases of different inflammatory dermatoses showed expression of both calgranulin chains in lesional but not in non-lesional skin. Keratinocytes in inflammatory dermatoses therefore express an intracellular calcium binding protein which has cytokine activity.

Topics: Antibodies, Monoclonal; Calcium-Binding Proteins; Calgranulin A; Calgranulin B; Dermatitis, Contact; Epidermis; Graft vs Host Disease; Humans; Keratins; Psoriasis; Skin Diseases, Vesiculobullous

HLA-DR expression by keratinocytes and enterocytes was studied in 23 patients undergoing BMT (12 autologous; 11 allogeneic). Two monoclonal antibodies were used to detect the HLA-DR antigen. Only in two patients before transplant and in one following autologous BMT was HLA-DR expressed on keratinocytes. Of 11 allogeneic recipients, 7 developed clinical GVHD, and HLA-DR-positive keratinocytes were seen in 6 of these. HLA-DR was expressed by enterocytes in 5 patients with GVHD and 4 of these also showed HLA-DR expression by keratinocytes. HLA-DR expression by keratinocytes correlated well with clinical GVHD. Expression of this antigen by enterocytes was associated with characteristic histological appearances of GVHD, even in the absence of intestinal symptoms. A combination of traditional and immunocytochemical techniques offers a sensitive and accurate method of confirming GVHD before it becomes florid.

Topics: Epidermal Cells; Epidermis; Epithelium; Graft vs Host Disease; HLA-D Antigens; Humans; Immunoenzyme Techniques; Keratins; Prospective Studies; Rectum

Recent evidence suggests that Ia+ Langerhans cells may be a primary target for destruction in cutaneous graft-versus-host disease (GVHD). Although it is generally accepted that T lymphocytes with helper/inducer phenotype are essential, the identity of the effector cells is still controversial. We therefore investigated whether a variety of Ia-reactive cloned helper T cells with different cross-reactivities and functions in vitro can induce cutaneous GVHD following intradermal inoculation into the footpad of the appropriate recipients, whose Ia antigens are able to stimulate the T cells to proliferate in vitro. All cloned T cells tested caused significant footpad swelling in their appropriate recipients with a course typical for local cutaneous delayed-type hypersensitivity (DTH) reactions. Two of these cloned T cells, SK 1 and BB5, induced local histologic changes consistent with grades 2-3 of cutaneous GVHD in the appropriate allogeneic or syngeneic recipients at 48-72 hr after their intradermal inoculation. Immunohistochemical studies using monoclonal antibodies demonstrated that not only injected cloned T cells but also Lyt-1+ cells derived from the recipient migrate into the epidermis and are responsible for the destruction seen in cutaneous GVHD. In epidermis in which cutaneous GVHD had been induced, expression of Ia by keratinocytes and the damage of Ia+LC were observed. These results suggest that Ia+LC and Ia+ keratinocytes may play an important role in the infiltration of Ia-reactive T cells responsible for cutaneous GVHD.

Topics: Animals; Clone Cells; Epidermal Cells; Epidermis; Female; Graft Rejection; Graft vs Host Disease; Histocompatibility Antigens Class II; Hypersensitivity, Delayed; Immunization, Passive; Keratins; Mice; Skin Transplantation; T-Lymphocytes, Helper-Inducer

Cutaneous graft-versus-host disease (GVHD) provides a unique model for studying the pathogenesis of several important lymphocyte-mediated skin diseases. Morphologic studies have suggested that Ia antigen (Ia)-bearing epidermal Langerhans cells (LC) may be specific targets for destruction in these conditions. Keratinocytes synthesize and express Ia in GVHD and some other lymphocyte-mediated skin disorders; Ia+ keratinocytes, constitutively able to secrete epidermal cell-derived thymocyte activating factor (ETAF)/interleukin 1, may possess antigen-presenting capacity, thus leading to enhanced cutaneous immune responses and disease chronicity. We therefore investigated the fate of Ia+ LC, and the potential antigen-presenting capacity of Ia+ keratinocytes, in a murine model of GVHD. Lethally irradiated C3H/He (H-2k) mice developed acute cutaneous GVHD, and expressed keratinocyte Iak, 8 days after injection of BALB/c (H-2d) bone marrow and spleen cells. Immunofluorescence studies showed a progressive decrease in the density of Ia+ epidermal LC during the evolution of GVHD. This decrease was paralleled by a progressive reduction in the allostimulatory capacity of GVHD epidermal cells (EC) in the allogeneic EC-lymphocyte reaction (ELR). The fall in the density of Ia+ LC, and in EC allostimulatory capacity in both primary and secondary ELRs, was consistently greater in GVHD mice than in mice treated only with x-irradiation. The allostimulatory capacity of GVHD and x-irradiated EC could not be restored by addition of indomethacin or exogenous ETAF to ELR cultures. The decreased allostimulatory capacity was not the result of inhibition of the ELR, since EC from GVHD and x-irradiated mice did not cause suppression when added to control ELR cultures. The capacity of EC to present ovalbumin, purified protein derivative of tuberculin, 2,4,6-trinitrobenzenesulfonic acid coupled to EC, and native cytochrome c (CYTc) to antigen-specific T-cell lines, clones, or hybridomas was reduced in x-irradiated mice and markedly decreased in GVHD mice. The capacity of EC from x-irradiated and GVHD mice to present CYTc fragment 81-104, which does not require further processing or catabolism by accessory cells, was similarly decreased. Taken together, the results indicate that: the function of LC is markedly and progressively impaired in acute GVHD; LC function is also decreased, but to a lesser extent, following x-irradiation alone; and Ia+ keratinocytes from lethally irradiated mice

Topics: Acute Disease; Animals; Antigen-Presenting Cells; Epidermis; Female; Fluorescent Antibody Technique; Graft vs Host Disease; Histocompatibility Antigens Class II; Isoantigens; Keratins; Langerhans Cells; Lymphocytes; Mice; Mice, Inbred Strains; Skin Diseases

As opposed to normal human skin where HLA-DR expression is restricted to the Langerhans cell (LC) population, HLA-DR, but not HLA-DS antigens can be readily detected on keratinocytes (KC) in certain disease states, i.e., cutaneous T cell lymphoma (CTCL), graft-vs-host disease (GVHD), and lichen planus (LP). To clarify the cellular origin of KC-bound HLA-DR antigens, we used a monoclonal antibody directed against determinants solely expressed on the cytoplasmic HLA-DR gamma chain (VIC-Y1) and observed that, by immunofluorescence, KC displaying HLA-DR alpha/beta complexes on their surface uniformly displayed cytoplasmic VIC-Y1 reactivity. In view of the crucial role of the gamma chain for HLA-DR biosynthesis, we conclude that HLA-DR antigens on KC are actively synthesized by these cells.

Topics: Epidermis; Fluorescent Antibody Technique; Graft vs Host Disease; Histocompatibility Antigens Class II; HLA-DR Antigens; Humans; Keratins; Lichen Planus; Lymphoma; Skin Diseases; T-Lymphocytes

Antibodies to Merkel cells (MC) are not known to occur in human sera. Using a newly described technic, we looked for the presence of antibodies directed against skin structures in sera from 2,300 patients with various dermatologic and hematologic disorders, as well as graft-versus-host disease (GVHD). Three of these sera (0.1%) were found to react with MC. One monoclonal IgM antibody was present in serum obtained from a patient with chronic cold agglutinin disease, the second from a patient with GVHD, and the third from a patient with pemphigus vulgaris. The present inadequate knowledge about normal MC function and the absence of described symptoms of MC dysfunction preclude analysis of the pathogenic significance of these MC antibodies in human sera.

Topics: Aged; Anemia, Hemolytic, Autoimmune; Animals; Antibodies, Monoclonal; Epithelial Cells; Female; Fluorescent Antibody Technique; Graft vs Host Disease; Humans; Immunoglobulin M; Keratins; Rabbits; Skin; Skin Diseases