Background. The heat shock proteins are increasingly becoming associated with immunopathologic phenomena, being induced in response to inflammation. They are highly immunogenic and are postulated as playing a role in both innate and adaptive immunity. Their proposed role in peptide binding and antigen presentation could suggest a potential role in the alloreactive process that leads to graft-versus-host disease (GVHD) after bone marrow transplantation.
Methods. In this study we examined the expression of the widely studied heat shock protein 70 (hsp70) in an in vitro-generated graft-versus-host reaction in human skin, using streptavidin biotin immunohistochemistry and laser scanning confocal microscopy.
Results. Hsp70 expression was correlated with high graft-versus-host responses (P <0.001) and was confirmed using laser scanning confocal microscopy. Increased expression of hsp70 was further defined due to increases in the inducible form of hsp70. Expression of inducible hsp70 was predictive of both clinical acute GVHD (P =0.001) and incidence of chronic GVHD (P <0.001).
Conclusions. This investigation has demonstrated for the first time the expression of hsp70 in a human model of GVHD, suggesting involvement in the pathogenesis of the disease and providing the basis for further investigation. Increased expression of inducible hsp70 in the model could provide a biologic marker for the prediction of clinical acute and chronic GVHD.
Graft-versus-host disease (GVHD) is a major limitation of bone marrow transplantation (BMT) and accounts for 25% of transplant-related mortality (1). Acute graft-versus-host disease (AGVHD) is a T cell-mediated disease affecting multiple target organs including the skin. The mechanism of the disease is complex and largely depends upon a histocompatibility antigenic difference between donor and recipient, involving both immunocompetent cells and cytokines, which can initiate apoptosis and necrosis of host tissue (2).
Since heat shock proteins (hsps) were first discovered (3) in 1962, they have been increasingly associated with immunopathologic phenomena, suggesting a possible role in the biology of GVHD. Hsps are evolutionarily conserved molecules that exist in virtually all mammalian, bacterial, and plant cells and that function as molecular chaperones to assist in protein folding, repair, and translocation (4). Increased synthesis of hsps can occur in response to a variety of stimuli including hyperthermia, glucose deprivation, and inflammation (5).
Hsps are characterized by molecular weight. Heat shock protein (hsp70) has two forms. Constitutive hsp70 is present in the cell at low levels and is not usually increased in situations of cellular stress, acting as a molecular chaperone within the normal cellular environment. Increased expression of inducible hsp70, on the other hand, occurs in response to stresses placed upon the cell (5).
Hsps are highly immunogenic, and it has been postulated that they could play a role in both innate and adaptive immunity, eliciting an immune response by acting as self-antigens expressed in a tissue-specific manner, containing regions that act as foreign epitopes, and presenting foreign antigens to the immune system (6).
Immune reactivity against hsps has been described in autoimmune disorders including rheumatoid arthritis (7) and multiple sclerosis (8). Studies postulate that exposure to microbial hsps can prevent immunologic tolerance by the stimulation of cross-reactive T cells that respond to hsp epitopes shared by both microbe and host, which can result in autoimmunity (5). Hsps have also been implicated in the stimulation and regulation of both proinflammatory and immunoregulatory cytokines (9,10).
Increases in hsp expression have been associated with transplant rejection in rejected skin grafts in wild-type mice (11) and in human kidney undergoing rejection (12). Elevated levels of hsp70 have also been observed in the lymph nodes and spleen of rats induced with acute lethal GVHD (13). More recently, antibodies to hsp70 have been associated with GVHD in peripheral blood stem-cell transplantation (14).
The human in vitro skin explant model has been successfully used for predicting AGVHD in HLA-matched sibling BMT (15,16). The model has also been used to investigate possible mechanisms involved in the pathogenesis of AGVHD (17), including the role of minor histocompatibility antigens (18). Recent data strongly suggest a target organ specificity of GVHD-related tissue damage and that the main target cells for GVHD are keratinocytes (19).
Our investigation aimed to correlate the expression of hsp70 with the severity of graft-versus-host reactions (GVHR) in skin sections generated from the human skin explant model. We compared the histopathologic evaluation of GVHR in patient skin biopsy specimens with the level of hsp expression using both immunohistochemistry and immunofluorescence. This investigation aimed to establish whether hsps could play a possible role in the pathogenesis of AGVHD and if they could provide an additional predictive indicator for the severity of clinical AGVHD and chronic graft-versus-host disease (CGVHD).