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Immunohistology of Stress Protein Expression during Chronic Rejection of Rat Cardiac Allografts

Part 1: Members of the Hsp70 Family

René J. Duquesnoy, Ph.D.

Professor of Pathology and Surgery

University of Pittsburgh Medical Center

Introduction

The pathogenesis of chronic rejection seems to involve a "response to injury" mechanism manifested as a continuous, low-grade inflammatory response initiated by alloimmune mechanisms. During this process, the allograft undergoes a stress response which alters the expression of heat shock proteins (hsp) and activates immune mechanisms involving hsp.

This concept has been tested with a rat cardiac allograft model of chronic rejection, developed recently by Murase and co-workers at the Thomas E. Starzl Transplantation Institute. Heterotopic heart transplants from fully histoincompatible Lewis rats into Brown Norway recipients pretreated 100 days before with Lewis bone marrow (BM) cells and a brief course of tacrolimus (FK-506), exhibit long-term graft survival without immunosuppression. All transplanted hearts in the BM group develop chronic rejection as evidenced by a chronic obliterative arteriopathy. On the other hand, recipients pretreated with a liver allograft (the OLTx group), do not develop chronic rejection of the subsequent cardiac allograft and this appears related to a hematolymphoid chimerism involving donor dendritic cells but the exact mechanisms have yet to be determined. In the BM group, a persistent immunological injury during the early post-transplant period is associated with the development of chronic rejection.

This chronic rejection model is characterized by an obliterative arteriopathy with fibrointimal hyperplasia and an accumulation of leukocytes in the intima and especially, the adventitia of arterial walls. There are also lymphocytic aggregates resembling Quilty lesions in the endocardium and epicardium. A recent sequential analysis of the changes that lead to chronic rejection has raised the concept that the intermittent disruption of the cardiac lymphatic flow due to chronic interstitial inflammation may represent a principal mechanism for development of the Quilty lesions and the pathogenesis of obliterative arteriopathy.

A recent review (click on HSP) summarizes the evidence that heat shock proteins are important in transplant immunity. Also called stress proteins, hsp are common constituents of all types of cells and they play a major role in various cellular compartments by functioning as molecular chaperones involved in the assembly, folding and translocation of intracellular polypeptides, to interact with various receptors and in stressed cells, to restore the functional activity of denatured proteins. Hsp mediate responses to a variety of stressful stimuli and they may increase the cellular resistance to injury. Many stress proteins have been classified as hsp according to molecular weight in kilodaltons. For instance, hsp60 operates primarily in mitochondria whereas the cytosolic compartment contains the stress-inducible hsp72 and the constitutively expressed hsp73 (also called hsc73). Other stress proteins are referred to as glucose-regulated proteins (grp) because they were originally discovered in culture systems deprived of glucose. For instance, Grp78 (also called BiP) is a molecular chaperone that operates primarily in the endoplasmic reticulum. Many stress proteins, especially the members of the hsp70 family, have a common structure consisting of an amino-terminal domain with ATPase activity and an carboxy-terminal domain with structural similarities to the peptide-binding region of MHC molecules.

Many investigators have demonstrated the involvement of hsp in autoimmune disease and in tumor immunity. Our studies on acute cardiac allograft rejection have indicated a role of heat shock proteins in transplant immunity and the reactivity of graft-infiltrating lymphocytes. Immunoblot analyses have shown increased expression of hsp60, hsp70 and grp78 and other stress proteins in stromal tissue preparations from acutely rejecting allografts. The proliferative responses of graft-infiltrating lymphocytes to allogeneic and syngeneic spleen cells are markedly augmented by mycobacterial hsp65 and especially hsp71. T-cell cloning experiments have permitted the identification autoreactive CD4-positive lymphocytes that respond to self-APC through mycobacterial hsp71-dependent or mammalian grp78-dependent mechanisms. Such hsp-dependent, autoreactive lymphocytes have also been isolated from cardiac allografts undergoing chronic rejection.

In this communication we report the tissue expression of hsp in Murase's model of chronic rejection. Part 1 of this article deals with members of the hsp70 family. The spectrum of stress proteins is extremely diverse and includes many other hsp including hsp27, hsp32 (also called hemoxygenase-1), hsp40, hsp47 (or colligin) and hsp60, protein disulfide isomerase and many others. Their immunohistology in chronic rejection will be described in subsequent parts of this report.

Materials and Methods

This report deals with the expression of various stress proteins in the transplanted hearts and the spleens from 100-day cardiac allograft recipients in the BM and OLTx groups and 100-day syngrafts as controls. The indirect avidin-biotin method was used for the immunostaining studies. All tissues were snap-frozen in optimal temperature compound and after storage at -70C, 4 uM cryostat sections were fixed in cold acetone at -20C. Heat shock protein expression was measured by immunostaining with monoclonal antibodies (mAbs) obtained from StressGen Biotechnologies Corp (Victoria, B.C., Canada). Part 1 of this report deals with the following mAbs reacting with rat hsp:

Monoclonal Antibody Specificity Intracellular Location
SPA-810 inducible form of hsp72 cytosol
SPA-820 inducible and constitutive forms of hsp70 cytosol
SPA-827 KSEKDEL peptide of grp78 (grp78seq) endoplasmic reticulum.

Immunoglobulin-matched monoclonal antibodies with irrelevant specificities (e.g. anti-HLA-A2) were used as negative controls for the hsp-specific monoclonals. A biotinated goat-anti-mouse Ig was used as the second antibody. The immunostaining was done by Dr Xiao-Fei Fu.

Results

Immunostaining with the grp78seq-specific antibody SPA-827

1. Transplanted hearts (Panel 1)

Please click on the picture for more details

1A

1B

1C

1D

1E

1F

1G

1H

1I

1J

1K

1L

1M

1N

1O

Panel 1 shows immunostaining results with the SPA-827 mAb generated against the synthetic peptide KSEKDEL sequence from rat grp78 conjugated to KLH (we call this peptide grp78seq). SPA-827 cross-reacts with grp78, grp94 and an undefined 50kDa KDEL-containing protein. In the BM group of heart allografts, clusters of grp78seq-positive cells intersperse between lymphocytes infiltrating the arterial walls and the interstitium (1A-1E ). The grp78seq staining pattern is granular (1D) and involves cells resembling dendritic cells. Previous studies by Demetris et al. have shown similar clusters of OX62-positive dendritic cells amid lymphoid aggregates in the adventitia of arteries with intimal thickening. Many grp78seq-positive cells are also seen between the mural surface and the endocardial and epicardial lymphocytic infiltrates (1F-1J) which, as previous studies have shown, consist of primarily CD4+ T-cells and CD8+ T-cells. Figures 1K and 1L show clusters of grp78seq-positive cells in a cardiac allograft friom BM-pre-treated recipients. No significant SPA-827 staining is seen for the infiltrating lymphocytes themselves, nor in the myocytes or fibrotic tissues. As previously described, heart allografts in the OLTx pre-treatment group exhibit much less lymphoid infiltration and no arteriopathy nor Quilty lesions. In the OLTx group, a few grp78seq-positive cells are seen in the myocardium and around blood vessels, and none are noted in the outer layers of the endocardium and epicardium (1M). Similarly, grp78seq expression is low in 100-day syngrafts and normal cardiac tissues (1N,1O).

2. Grp78seq staining in spleens (Panel 2)

2A

2B

2C

2D

2E

2F

2G

2H

2I

2J

The most prominent feature of the immunostaining with SPA-827 is the presence of many grp78seq-positive cells dispersed between the sinuses in the red pulp and, especially in the BM-pretreated group of allograft recipients, in the marginal zones of the periarterial lymphatic sheaths in the white pulp (2A-2F). The marginal zone is characterized by a reticular network into which enter many arterial branches of the central artery as well as venous sinuses. This area is rich in dendritic macrophages and lymphocytes and represents a major site for antigen uptake. Intravenously administered particulate material such as carbon, or labeled soluble antigen is initially dispersed in the red pulp and then concentrated in the marginal zones of the spleen. Circulating T-cells and B-cells enter the splenic white pulp by transversing the vascular sinuses in the marginal zones. Thus, the marginal zones seem to represent a location in the splenic white pulp where T-cell and antigen-presenting cells first encounter

Grp78seq-positive cells have the morphological appearance of macrophages rather than lymphocytes and they may represent a population of antigen-presenting cells. Their presence in the marginal zones is most pronounced in the BM group (2A-2F) followed by the OLTx group (2G,2H) and to a lesser extent, in 100-day syngraft recipients (2I) and normal non-transplanted hearts (2J). Thus during chronic rejection, grp78seq-positive cells might interact with T-cells in the marginal zones and this may lead to subsequent T-cell activation in the periarterial lymphatic sheaths.

Staining with hsp70-specific mAbs SPA-810 and SPA-820

Transplanted hearts (Panel 3)

3A

3B

3C

3D

3E

3F

3G

3H

3I

3J

3K

3L

SPA-820 is an hsp70-specific mAb that reacts with both inducible hsp72 and constitutive heat shock cognate 73 or hsc73. Its immunostaining pattern is very different from that seen with the grp78pep-specific SPA-827 mAb. In the BM group of heart allografts, no or few hsp70-positive graft-infiltrating cells are seen around affected arterial blood vessels (3A-3D). On the other hand, the arterial walls themselves show often intense staining of hsp70 (3A, 3B), especially in the areas of intraluminal myofibroblast proliferation. Also, only a few hsp70-positive cells infiltrate the Quilty lesions (3D-3F), whereas many underlying myocytes show increased hsp70 expression as compared to myocytes in areas without cellular infiltration.

SPA-820-positive infiltrating cells can be seen in some inflammatory lesions (3G, 3I), but not in others (3J). The intercalated disks of cardiac myocytes showed readily detectable expression of hsp70 in the BM group (3H, 3G) equally well as in the OLTx group (3K) and in normal hearts (3L).

The mAb SPA-810 specific for hsp72, the heat stress inducible form of hsp70, shows no significant staining in any allograft, syngraft or normal heart tissue examined in this study (data not shown).

Hsp70 staining in spleens (Panel 4)

4A

4B

4C

4D

4E

4F

4G

4H

In the splenic follicles, the central arterioles and their branches are readily visualized by the hsp72+hsc73-specific mAb SPA-820 and the intensity of this concentric staining pattern (4D) is stronger in the allograft recipients in the BM and OLTx pre-treatment groups than in spleens from syngraft recipients (4F) and normal rats (4G). Many SPA-820 positive cells with rod-like staining patterns are present in the red pulp and to a much lesser extent, in the splenic follicles (4A-4G). Their morphology and distribution is different from that of grp78seq-positive cells.

Except for some vessel walls, there was little staining of splenic tissue with SPA-810 specific for the heat-inducible hsp72 (4H).

Conclusions

In summary, these immunostaining results demonstrate different expression patterns of the members of the hsp70 family in cardiac allografts undergoing rejection as compared to non-rejecting allografts, syngrafts and normal hearts. Most salient findings are the clusters of grp78seq-positive graft-infiltrating cells and the strong staining of the constitutively expressed hsc73 in obliterating arteries. During chronic rejection, hsc73 expression is also higher in allograft myocytes especially near areas of cellular infiltration.. No allograft staining was seen with antibodies against the inducible hsp72 and negative controls such as anti-HLA-A2.

Grp78seq-positive cells are readily found in the red pulp of the spleen and in the allograft recipients, especially those with chronic rejection, they appear to localize in the marginal zones of the T-cell dependent periarterial lymphatic sheaths. The red pulp contains also hsc73-positive cells and their numbers appear to be similar in allograft and syngraft recipients and normal controls.

Selected References

Demetris AJ, Murase N, Lee R, et al. Analysis of Chronic Rejection and Obliterative Arteriopathy. Possible Contributions of Donor Antigen Presenting Cells and Lymphatic Disruption. Amer. J. Pathol. 1997; 150: 563

Murase N, Starzl TE, Tanabe M, et al. Variable chimerism, graft-versus-host disease, and tolerance after different kinds of cell and whole organ transplantation from Lewis to brown Norway rats. Transplantation 1995; 60: 158.

Duquesnoy R, Liu K, Moliterno R, Attfield: D. Do heat shock proteins play a role in transplant immunity? Transplantation Reviews 1996; 10: 175.

Qian J, Moliterno R, Donovan-Peluso M, et al. Expression of heat shock proteins and lymphocyte reactivity in rat cardiac allografts undergoing cellular rejection. Transplant Immunol 1995; 3: 114.

Moliterno R, Valdivia L, Pan F, Duquesnoy RJ. Heat shock protein reactivity of lymphocytes isolated from heterotopic rat cardiac allografts. Transplantation 1995; 59: 598.

Liu K, Moliterno R, Fu X-F, Duquesnoy: R. Identification of two types of autoreactive T lymphocyte clones cultured from rat cardiac allograft cells incubated with recombinant mycobacterial heat shock protein 71. Transplant Immunol 1996;5:57.

 


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