Sarcolemmal Complement Membrane Attack Complex Deposits During Acute Rejection of Myofibers in Nonhuman Primates

Abstract

We have previously studied in nonhuman primates several aspects of the acute rejection of myofibers, including the histological characteristics, the mechanisms of myofiber elimination by the T cells, and the development of anti-donor antibodies. Here, we report the participation of the complement membrane attack complex (MAC) in this context. We used muscle sections of macaques from experiments of allogeneic muscle precursor cell transplantation with confirmed rejection of the graft-derived myofibers. Sections were stained with hematoxylin and eosin, alizarin red and for immunodetection of MAC, CD8, CD4, C3, C4d, and immunoglobulins. The prominent finding was the presence of sarcolemmal MAC (sMAC) deposits in biopsies with ongoing acute rejection or with recent acute rejection. The numbers of sMAC-positive myofibers were variable, being higher when there was an intense lymphocyte infiltration. Few sMAC-positive myofibers were necrotic or had evidence of sarcolemma permeation. The immunodetection of C3, C4d, and immunoglobulins did not provide significant elements. In conclusion, sMAC deposits were related to myofiber rejection. The fact that the vast majority of sMAC-positive myofibers had no signs of necrosis or sarcolemmal permeation suggests that MAC would not be harmful to myofibers by itself.

FIGURE 1.

Different aspects linked to sarcolemmal membrane attack complex (sMAC) detection in cell-grafted muscles during acute rejection of myofibers. (A–C) Serial cross-sections (monkey IW-3, 8 weeks after tacrolimus withdrawal) processed for fluorescent immunodetection of C5b-9 (A) and CD8 (C), and stained with hematoxylin and eosin (H&E) (B). They are shown at low magnification to illustrate the abundance of sMAC-positive myofibers (A), focal lymphocyte accumulations (B, dark basophilic spots), and CD8-positive cells (C, the inset is a magnification of the indicated region). The regions in the rectangles in A and B are respectively enlarged in D and E to better see the sMAC-positive myofibers and the topographic correlation with the lymphocyte accumulations. The intensity of sMAC labeling is variable, and myofibers with more intense sMAC labeling are essentially close or within the lymphocyte accumulations. (F) At higher magnification, the myofibers indicated by arrowheads in D and E illustrate that they are sMAC-positive (D) but have a preserved intermyofibrillar network (F). The “a” arrow in D and E indicates a sMAC-positive myofiber that exhibits the classical aspect of lymphocyte invasion of non-necrotic myofibers (the intermyofibrillar network is preserved, as can be seen in the inset in E) typical of the T lymphocyte attack on myofibers. The “b” arrow in D and E points to a myofiber with lymphocyte invasion that is not clearly sMAC-positive in the serial section. (G, H) Serial cross-sections (monkey IW-3, 10 weeks after tacrolimus withdrawal) stained with alizarin red ([AR] G) and processed for fluorescent immunodetection of C5b-9 (H). The arrows point to 2 AR-positive myofibers that are sMAC-positive in the serial section, while the arrowheads point to AR-positive myofibers that have no sMAC. Some sMAC-positive myofibers are not stained with AR (which illustrate the AR reaction in the majority of the sMAC-positive myofibers [asterisks]). Scale bars: A–C = 1 mm; D, E, G, H = 100 µm; F = 25 µm.

FIGURE 2.

Correlation of sarcolemmal membrane attack complex (sMAC) with other transplantation parameters in the monkeys of the IW (immunosuppression withdrawal) protocol. Graphs for each monkey (in columns) display the number of sMAC-positive myofibers (red graphs: number of sMAC-positive myofibers per mm² of grafted muscle in the biopsy), graft-specific labels (blue graphs: percentage of the sectional area of the muscle that was β-Gal-positive; green graphs: density of the Y chromosome PCR band in ImageG units), and lymphocyte accumulations (violet graphs: percentage of the grafted area in the biopsy occupied by lymphocyte accumulations). The arrows designated as “sampling” at the top indicate the moment of both muscle biopsies and blood samples for detection of anti-donor cell antibodies (ADCA). X-axes indicate weeks after tacrolimus withdrawal. Orange bars in the top show the period in which de novo circulating ADCA were detected. Both the peak of sMAC-positive myofibers and the peak of lymphocyte infiltration concur with the abrupt decrease and/or the disappearance of the grafted-cell labels. In the red graphs, the periods in which the MAC labeling was slight and more frequently partial are lighter than the periods in which MAC labeling was intense and more frequently complete around the myofiber contour.

FIGURE 3.

Sarcolemmal membrane attack complex (sMAC) in the LI (low immunosuppression) protocol. (A) Panel illustrates the correlation of sMAC with other parameters, similarly and at similar scale as in Figure 2: sMAC-positive myofibers per mm² of grafted muscle (red graphs), as well as the percentages of the sectional area of the cell-grafted muscle that were β-Gal-positive (blue graphs) or occupied by lymphocyte accumulations (violet graphs). (B–D) Serial cross-sections of a cell-grafted muscle region in monkey LI-2, 4 weeks post-transplantation, stained for fluorescent immunodetection of C5b-9 (B), for β-Gal demonstration (C), and with hematoxylin and eosin (H&E) (D). This biopsy shows the correlation between β-Gal-positive myofibers and sMAC. All sMAC-positive myofibers are β-Gal-positive (some marked with asterisks). However, most β-Gal-positive myofibers have no sMAC, as seen in the regions indicated by arrowheads. An accumulation of lymphocytes (arrows in B–D) surrounds β-Gal-positive myofibers, most of which are sMAC-positive. Myofibers close to this lymphocyte accumulation, as in other cases, are those with the most intense sMAC labeling. Scale bars = 100 µm.

FIGURE 4.

Co-immunodetection of C5b-9 (green fluorescence) and CD8 (red fluorescence) in cross sections of a biopsy taken in a cell-grafted site at the peak of lymphocyte infiltration using confocal microscopy. Some myofibers (asterisks) present linear sarcolemmal membrane attack complex (sMAC) deposits covering completely, or partially, the myofiber contour. Two necrotic myofibers (A, D, arrows) are identified by the sarcoplasmic MAC labeling, which indicates damage of the sarcolemma allowing penetration of complement that was activated and formed intracellular MAC deposits. (A) The necrotic myofiber has also sMAC (arrowhead). (D) No sMAC is discernible in the necrotic myofiber. Most of the sMAC-positive myofibers in the images are in contact with CD8-positive lymphocytes. (D–F) The myofiber with sarcoplasmic MAC deposition is not surrounded by CD8-positive lymphocytes, which may be due to the fact that it is already in an advanced stage of necrosis (as the intense intracellular MAC labeling suggests) and was possibly left aside by the CD8-positive lymphocytes. Scale bars = 50 µm.