Peter C. Wever

Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection

BACKGROUNDnGraft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC.nnnMETHODSnExpression of perforin, GrB and FasL was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies. Specific inhibitors of the perforin/GrB- and FasL-based pathways were used in 51Cr release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines.nnnRESULTSnCells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-FasL polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC. Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas.nnnCONCLUSIONSnThe perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.

Quantification of Bax/Bcl-2 ratios in peripheral blood lymphocytes, monocytes and granulocytes and their relation to susceptibility to anti-Fas (anti-CD95)-induced apoptosis

Neutrophils have the shortest half‐life among circulating leucocytes and rapidly undergo apoptosis in vitro. The homologous Bcl‐2 and Bax proteins have opposing effects, with Bcl‐2 extending cellular survival and Bax promoting cell death following an apoptotic stimulus. We determined Bcl‐2 to Bax expression ratios in peripheral blood lymphocytes, monocytes and granulocytes and related them to the susceptibility of these cells to anti‐Fas (anti‐CD95)‐induced apoptosis. Here, we show that Bax/Bcl‐2 ratios are high in granulocytes and relatively low in monocytes and lymphocytes. Furthermore, we show a relation between this ratio in the different leucocyte subsets and their susceptibility to anti‐Fas‐induced apoptosis, with granulocytes showing the highest susceptibility, followed by monocytes and lymphocytes. It is concluded that the balance between Bcl‐2 and Bax forms an apoptotic rheostat, which seems to determine sensitivity to apoptosis.

Apoptosis of acinar cells in pancreas allograft rejection

BACKGROUNDnRecently it has been recognized that apoptosis of target cells may occur during liver and kidney allograft rejection and is probably induced by infiltrating cells. Pancreas rejection is also characterized by a cellular infiltrate, however, the occurrence of apoptosis has not been investigated. We assessed whether pancreas rejection was associated with apoptosis of target cells and an influx of granzyme B (GrB)-positive or CD68-positive cells.nnnMETHODSnEighteen pancreas biopsies (10 of 18 with rejection) from 15 patients with a pancreas-kidney transplantation were stained with the in situ end-labeling method for apoptosis, and for CD3, GrB, and CD68.nnnRESULTSnSignificantly more apoptotic acinar cells were found in biopsies with rejection when compared with biopsies without rejection. No difference was observed between the groups for GrB+ or CD68+ cells.nnnCONCLUSIONnWe conclude that pancreas rejection is associated with apoptosis of acinar cells, but not with an increased influx of GrB+ cells or macrophages.

The CD8+ granzyme B+ T‐cell subset in peripheral blood from healthy individuals contains activated and apoptosis‐prone cells

Granzyme B (GrB) has been implicated in induction of apoptosis in target cells. The presence of GrB in peripheral blood CD8+ Tu2003cells from healthy individuals was analysed in immunocytochemical and flow cytometric studies. Furthermore, CD8+ GrB− Tu2003cells and CD8+ GrB+ Tu2003cells were compared regarding phenotypical characteristics and susceptibility to both spontaneous and Fas‐mediated apoptosis. GrB was expressed by approximately one‐fifth of CD8+ Tu2003cells. Compared with the CD8+ GrB− T‐cell subset, the CD8+ GrB+ T‐cell subset contained cells that were relatively more activated and more prone to spontaneous apoptosis. Culturing of cells with immunoglobulin M (IgM) anti‐Fas monoclonal antibody had no additional effect on the number of CD8+ GrB+ Tu2003cells undergoing apoptosis. We suggest that the presence of CD8+ GrB+ Tu2003cells in peripheral blood from healthy individuals results from immune surveillance or contact with infectious agents, and that spontaneous apoptosis of these cells might serve as a mechanism for their eventual clearance.

Immunocytochemical detection of granzymes A and B in peripheral blood lymphocytes from healthy individuals after non-enzymatic antigen retrieval

Cytoplasmic granules of cytotoxic lymphocytes contain several constituents, including perforin, granzyme A (GrA), and granzyme B (GrB). After granule exocytosis, GrA and GrB are believed to enter the target cell through perforin-derived transmembrane channels to induce DNA fragmentation and apoptosis (Liu et al. 1995; Nakajima et al. 1995; Smyth and Trapani 1995; Heusel et al. 1994). Various reports have dealt with expression of GrA and GrB in peripheral blood lymphocytes from healthy individuals, but the findings are conflicting (Berthou et al. 1995; Kummer et al. 1995; Sunder–Plassmann et al. 1990). We addressed this matter through the application in immunocytochemistry of antigen retrieval methods routinely used in immunohistochemistry. Cytocentrifuge preparations of lymphokine-activated killer (LAK) cells were fixed in 4% buffered formalin for 10 min. Part of the preparations were left untreated, and either enzymatic or non-enzymatic antigen retrieval was employed on the other part. Two enzymatic antigen retrieval methods were applied: digestion with 0.1 mg/ml pepsin A in 10 mM HCl, pH 2.0, at 37C, and digestion with 0.1 mg/ml protease Type XIV in PBS, pH 7.4, at 37C. In both cases, digestion was stopped after either 30 sec, 1 min, 2 min, or 4 min. Non-enzymatic antigen retrieval consisted of boiling the preparations in 10 mM sodium citrate, pH 6.0, for 10 min. Single-color stainings were performed with monoclonal antibodies (MAbs) GrA-6 and GrB-7, raised against recombinant human GrA and GrB proteins, respectively, which recognize GrA and GrB in paraffin-embedded, formalin-fixed tissue sections (Sanbio/ Monosan; Uden, The Netherlands). No staining was observed in the majority of untreated LAK cells (Figure 1A). Enzymatic antigen retrieval did not have a beneficial effect, and extension of digestion time, especially in the pepsin A-containing antigen retrieval solution, led to loss of cell number and loss of morphology (not shown). After non-enzymatic antigen retrieval, the majority of LAK cells displayed a strong granular staining pattern, but cell loss and loss of morphology were not observed (Figure 1B). The mechanisms underlying the effects of enzymatic and non-enzymatic antigen retrieval differ, and a selective benefit from one treatment over the other has also been demonstrated in immunohistochemistry for other antigens (Cattoretti et al. 1993). The effect of enzymatic antigen retrieval is known to depend on proteolytic restoration of accessibility of antibodies to masked antigens. Masking of antigens is an artifact induced by formalin fixation and is believed to be due to formation of methylene crosslinks between reactive sites on proteins. The amount of crosslinking determines the ease with which high molecular weight antibodies diffuse towards their antigens. In contrast, the effect of non-enzymatic antigen retrieval appears to depend on protein denaturation, with heat accounting for most of this effect (Cattoretti et al. 1993). MAbs GrA-6 and GrB-7 were raised against recombinant granzymes produced in a prokaryotic expression system, and the fact that reactivity of these MAbs is enhanced after non-enzymatic antigen retrieval suggests that they specifically recognize granzymes with a denatured conformation. Several factors can be pointed out that might have induced conformational changes in the recombinant granzymes and may have contributed to this specificity, e.g., incorrect folding of the recombinant granzymes in the prokaryotic expression system or partial degeneration of the recombinant granzymes after immunization. When we extended fixation time to 7 days, we still did not observe an influence on staining results when we employed non-enzymatic antigen retrieval (not shown). This observation implies that use of MAbs GrA-6 and GrB-7 in immunocytochemistry is not hampered by masking of the respective antigens due to formalin fixation, and explains the lack of beneficial effect of enzymatic antigen retrieval. Subsequently, non-enzymatic antigen retrieval was performed on formalin-fixed cytocentrifuge preparations of peripheral blood mononuclear cells from 10 healthy individuals. Two-color stainings were performed combining polyclonal rabbit antihuman CD3 antibodies with MAb GrA-6 or GrB-7. Two observers independently scored 300 staining cells per slide. Cells staining doubly positive were clearly distinguishable from cells staining singly positive (Figure 1C). Identical staining patterns were observed for MAbs GrA-6

Interferon-γ preferentially reduces memory/effector CD8 T lymphocytes in healthy subjects

To evaluate the influence of interferon-gamma (IFN-gamma) on leukocyte dynamics, with a focus on naive and memory T cells, we studied 6 healthy subjects twice in a placebo-controlled trial: once after the administration of recombinant human IFN-gamma (rhIFN-gamma; 100 microg/m2 subcutaneously) and at least 4 weeks later, after the administration of saline solution. Additionally, we studied the expression of adhesion molecules on T lymphocytes after in vitro incubation of whole blood with rhIFN-gamma. IFN-gamma induced a significant depletion in the number of T lymphocytes (P <.05 vs control), which was more severe in the CD8+ cell subset than in the CD4+ T cell subset. The numbers of naive CD4+ T cells and memory CD4+ T cells were equally affected by IFN-gamma, whereas within the CD8+ T cell subset, memory/effector cells disappeared preferentially as compared with naive cells (P <.05 vs control). In addition, IFN-gamma induced a decrease in B cells, NK cells, and monocytes. After an initial increase, granulocyte counts decreased significantly as compared with controls. These effects appeared not to be caused by the minimal rise in plasma cortisol levels (P <.05 vs control). In vitro, IFN-gamma did not up-regulate the expression of CD11a, NKI L16, CD11b, LFA-3, or VLA-4. We conclude that the administration of a single dose of IFN-gamma to healthy subjects profoundly affects the numbers of several leukocyte subsets in the peripheral blood compartment

Increased numbers of granzyme-B-expressing cytotoxic T-lymphocytes in the small intestine of HIV-infected patients

The objective of this study was to determine whether granzyme B-expressing cells, which identify activated cytotoxic lymphocytes, are present in the small intestinal mucosa of human immunodeficiency virus (HIV)-infected patients with and without diarrhea. Therefore, duodenal biopsy specimens from 29 HIV-infected patients (11 with diarrhea and 18 without diarrhea) and 15 control patients were stained for the presence of granzyme B expressing cells. In HIV-infected patients, a significantly increased expression of granzyme B in the lamina propria was observed (p = 0.00001): In 22 of 29 patients, at least 5-10 cells per high-power field were counted. In contrast, in 13 of 15 control patients, granzyme B was not expressed or minimally so, and in two others a maximum of five granzyme-B-expressing cells could be detected per high-power field. No significant difference was found between the HIV-infected patients with and without diarrhea. Double staining revealed that the granzyme-B-expressing cells were mainly CD3 positive. These data show that activated cytotoxic T lymphocytes (CTLs) are present in the duodenal mucosa of HIV-infected patients. No relation between the number of CTLs and the presence of diarrhea was demonstrated. CTLs are known to be involved in the pathogenesis of HIV infection and in the production of tissue injury, but their functional role in intestinal HIV-related pathology has yet to be elucidated.