Springer Semin Immunopathol (2003) 25:141–165 DOI 10.1007/s00281-003-0134-2 © Springer-Verlag 2003 Immunopathology of organ transplantation Lorraine C. Racusen Department of Pathology, Carnegie 4, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287, USA Abstract.&p.1: Immunopathology techniques are useful and sometimes indispensable tools to diagnose and/or define pathogenesis of disease processes in an organ allo- graft. Using immunostaining and/or in situ hybridization, cells and molecules can be identified and localized in organ grafts. Considered in this chapter are the “priming” of tissues by ischemia and infection, cell-mediated and antibody-mediated acute and chronic rejection in xenografts and allografts, graft infection, and recurrent or de novo immune disease in organ grafts. The chapter is designed to provide an over- view, with illustrative examples of the contribution of immunopathology to analysis of solid organ allografts.&bdy: Introduction Immunopathology has contributed and continues to contribute greatly to diagnosis and to definition of pathogenesis of important pathological processes in solid organ allografts. These processes include ischemia/reperfusion injury with tissue “prim- ing”, allograft rejection, infection, and recurrent disease in the allograft. Immunopa- thology is useful in confirming the presence of acute and chronic cell-mediated re- jection, and is particularly crucial for tissue diagnosis of antibody-mediated rejec- tion, each of which will be considered in this chapter. The immunopathology of both xenografts and allografts will be discussed. Technical aspects of immunostaining will be outlined briefly as well. Techniques Immunopathology is a broad term encompassing immunohistochemistry, immuno- fluorescence and in situ hybridization. Broadly, antibodies or probes specific for tar- get proteins or nucleic acids are reacted with tissue sections and attach to the target antigen. The type of tissue that can be utilized for such studies depends on the tech- nique, the stability of the antigen, and the avidity of the probe. For detection of some antigens, staining must be performed on frozen tissue. Other protein antigens and nu- 142 L.C. Racusen cleic acid sequences can generally be detected in formalin-fixed paraffin-embedded tissue. Some preparation of the tissue, such as protease digestion or microwaving, may be required to “unmask” the antigen; these techniques must be carefully con- trolled and standardized, to avoid over-interpretation of staining. Intracellular mole- cules such as cytokines cannot be detected reliably in formalin-fixed tissue, but can be detected by immunostaining of tissue fixed in acetone. In immunohistochemistry, the antibody or probe carries an enzyme (e.g., peroxi- dase) which can trigger a chemical reaction, resulting in localized precipitation of a colored reaction product at the site of antigen that is detectable by light microscopy. For direct immunofluorescence, the antibody carries a dye that can be activated by UV light (e.g., fluorescein, rhodamine). In indirect immunofluorescence, the primary antibody specific for the antigen is not labeled, but tissue sections are reacted with a second labeled antibody to the primary antibody. With appropriate positive and nega- tive controls run concurrently, very precise detection and localization can be achieved. Intensity and extent of staining can be quantitatively assessed. In in situ hybridization, a labeled riboprobe is utilized that is complementary to a unique nucleic acid sequence in target cells or organisms. With a radio-labeled probe, following reaction of the probe with the tissue, sections are dipped in nuclear emulsion and exposed in the dark; silver grains from the emulsion precipitate at the site of the radio-label. Probes can also be labeled with digoxigenin; the probe can be detected using enzyme-labeled anti-digoxigenin antibody, with amplification to in- tensify precipitated reaction product. This technique also allows precise localization and can be quantitatively assessed. Relative localization of several different target antigens in a specimen can be as- sessed by staining of immediately sequential sections. However, the availability of a variety of colorimetric histochemical reactions and UV-activated dyes now allows detection of several different target antigens simultaneously on a single tissue sec- tion, which can provide very precise information about spatial relationships, with overlapping signals suggesting intimate interactions between antigens. A variety of different techniques for such multiple labeling are available (e.g., [147]). Recent ad- vances in immunostaining including new reporter molecules and methods to increase sensitivity and improve multiple immunostaining have been recently reviewed [146]. Recent advances in microscopy have further advanced the field. Confocal micro- scopy can be used to refine three-dimensional (3D) localization of labeled molecules. Quantitative digital fluorescence microscopy using laser scanning (thick sections) or wide-field microscopy (thin sections) allows collection of large data sets for molecu- lar localization in cells and tissues [6]. Different wavelengths for activation have en- abled deeper tissue penetration for 3D imaging in very localized volumes [29]. De- velopment of fluorescent tagging with green fluorescent protein can be used to study protein mobility in living cells [119]. These technologies have yet to be applied on any scale to studies of allograft biology, but doubtless will be applied to the field in the near future. While the focus of this review is primarily on immunopathology as applied to tis- sue sections, the same techniques can be and have been used to study material ob- tained via less invasive methods. Specimens obtained via fine needle aspiration of organs, bronchoalveolar lavage or collection of urine sediment, for example, can be examined for expression of phenotypic markers, markers signaling activation and proliferation, or “stress response”. Indeed, immunohistology of allograft aspirates may contribute significantly to specificity of diagnosis [120]. Suspended cells can be Immunopathology 143 exposed to one or more labeled antibodies and examined by light microscopy. With adequate numbers of cells, flow cytometry can be employed and cells analyzed by cell sorting. With appropriate purification and positive and negative controls, excel- lent characterization and quantitation of cells by phenotype is possible. Large num- bers of available dyes and technological advances have made this an increasingly useful technology (see [59] for recent review). Tissue priming/“Innate” immunity It is almost impossible to avoid injury to an organ allograft. Ischemia/reperfusion in- jury, supervening infection and other processes can trigger nonspecific innate im- mune and inflammatory responses in the graft. These injury responses in turn can trigger and amplify antigen-specific adaptive immune responses. This tissue “prim- ing” may lead not just to acute alloimmune reaction to the graft [84], but may precip- itate indolent chronic processes, eventuating in fibrosing changes in the allograft [50]. These processes have particular relevance in organs from cadaveric donors [8]. Immunohistology is being utilized as a tool to understand this “priming” process, and in kidney allografts, at least, may be useful in identifying recipients at particular risk for early graft dysfunction [130]. A number of investigators have used immuno- histology to demonstrate increased expression of adhesion molecules, HLA antigens (class II), and complement in both native and allograft organs which have undergone ischemia/reperfusion [42, 68, 136]. Infection, and especially cytomegalovirus infec- tion, may also produce analogous tissue changes [151]. Adhesion molecules contributing to homing and targeting of inflammatory infil- trates include intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and selectins. In experimental models, adhesion molecule ex- pression is altered in ischemia/reperfusion injury (reviews in [77, 142]). Analogous changes have been demonstrated in clinical renal biopsy specimens studied by im- munohistochemistry. For example, expression of ICAM-1 and VCAM-1 have been shown to be elevated in pre-transplant donor biopsy samples from cadaveric com- pared to live donor kidney allografts; expression of ICAM-1 on tubular cells appears to be a predictor for delayed graft function, although not rejection, in cadaveric kid- neys [130]. Adhesion molecule expression, including ICAM, VCAM and E-selectin, have also been shown to be increased in cardiac allograft biopsy material post-trans- plant [19, 57]; in one study these changes were coincident with elevated serum tro- ponin T levels, indicative of ischemia [57]. New or enhanced expression of HLA antigens has been demonstrated by immu- nohistochemistry in organs with ischemic injury. In particular, HLA class II antigens, which are constitutively expressed on a limited number of cell types, may be ex- pressed by parenchymal cells following tissue injury. Class II DR expression has also been shown to be predictive of later allograft function [38]. Using immunohis- tology in sequential studies of unilateral kidney ischemia, Ibrahim et al. [68] were able to demonstrate increased expression of class II antigens and localize expression to tubular epithelial cells, peritubular capillaries and interstitial cells; this