The extent of peritubular CD14 staining in renal allografts as an independent immunohistological marker for acute rejection.

Previously, we demonstrated that in acute interstitial rejection, immunohistological staining of renal allograft biopsies with the CD14 mAb WT14, reacting with human monocytes/macrophages, shows a characteristic peritubular increase of positive cells. To test the diagnostic value of this CD14 positivity, we compared, in 154 unselected renal allograft biopsies, the extent of peritubular WT14 staining with (a) the original histological diagnosis, made with knowledge of clinical data, (b) the retrospectively and blindly scored histological diagnosis according to the criteria of the Banff classification, and (c) the eventual clinical diagnosis, which included evaluation of the response to therapy. The extent of peritubular WT14 positivity, blindly scored on cryostat sections of the frozen part of the biopsies, correlated positively with the probability of acute rejection (AR). When using a cutoff of 70% WT14 positivity for the diagnosis of AR, as extracted from a receiver operating characteristic curve, the WT14 diagnosis had a positive predictive value of 91% and a negative predictive value of 56%, compared with the original histological diagnosis. Compared with the Banff diagnosis of AR (grade I-III), these values were 95% and 47%, and compared with the clinical diagnosis, 84% and 63%, respectively. The WT14 diagnosis essentially corrected the original histological diagnosis in 7 cases, and was consistent with the eventual diagnosis in 5 equivocal cases. We conclude that the extent of peritubular CD14 positivity can be used as a marker for AR and can serve as a valuable additional criterion for AR in the histological examination of renal allograft biopsies.

Detection of interstitial increase in macrophages, characteristic of acute interstitial rejection, in routinely processed renal allograft biopsies using the monoclonal antibody KP1.

Acute interstitial rejection (AIR) of renal allografts is accompanied by a characteristic peritubular increase in macrophages, which can be identified with the CD14 monoclonal antibody (mAb) WT14 in cryostat sections. Since frozen tissue is not always available, we tested whether this increase can also be demonstrated in Bouin-fixed, paraffin-embedded biopsies, using the CD68 antimacrophage mAb KP1, which can also be applied to paraffin sections. Sections of 16 biopsies with AIR and 11 controls were stained with KP1. In 25 of the 27 biopsies, macrophages were strongly positive for KP1. Two AIR biopsies were completely negative, probably due to prolonged fixation. In the remaining 14 AIR biopsies, the number of KP1-positive cells was significantly higher than in the controls [1184 +/- 410 per mm2 (mean +/- SD) vs 112 +/- 126 per mm2]. We conclude that, especially in cases in which frozen tissue is not available, the demonstration of increased numbers of monocytes/macrophages with mAb KP1 can be a helpful adjunct in the histological diagnosis of AIR in routinely processed renal biopsies.

Immunocytology of urinary sediments as a method of differentiating acute rejection from other causes of declining renal graft function.

We have previously reported that during acute rejection of renal allografts T lymphocytosis and increased HLA-DR expression on tubular epithelial cells can be demonstrated in urinary sediments by incubating cytospin preparations with monoclonal antibodies against T cells and HLA-DR antigen in an indirect alkaline phosphatase technique. We now tested whether immunocytological analysis of urinary sediments can be used to differentiate acute rejection from other causes of declining graft function. For this we retrospectively selected, from a series of urinary samples that were taken either at random or as part of a longitudinal study in unselected graft recipients, those specimens that were taken at the time of increasing creatinine levels, and compared the original immunocytological diagnosis, made without knowledge of clinical data, with the final clinical one. In 44 of 74 evaluable cases an immunocytological diagnosis of rejection was made, which in 37 patients was consistent with the eventual clinical diagnosis. In 28 of 30 cases the diagnosis no rejection proved to be correct. This indicates a sensitivity of 95% and a specificity of 80% for the immunocytological diagnosis of rejection. Of 38 patients who underwent a renal core biopsy, the immunocytological diagnosis was consistent with the histological diagnosis in 36 cases (31 rejections, 5 no rejections). In this subgroup the sensitivity of the immunocytology was 97% and the specificity 83%. We conclude that immunocytological examination of urinary sediments in renal allograft recipients can be a valuable new tool in discriminating acute interstitial rejection from other causes of deteriorating graft function.

Variable expression of Ia antigens on the vascular endothelium of mouse skin allografts.

Ia antigens are membrane-bound glycoproteins that play a part in antigen recognition and subsequent cell-cell interactions in the immune response. In the mouse they are coded for by the I region of the major histocompatibility complex H-2 and have been demonstrated on B lymphocytes, monocytes, activated T cells, macrophages and dendritic cells, including Langerhans cells. Ia-like antigens have also been detected on the vascular endothelium in man and on epidermal keratinocytes in rats but expression on the latter cells was induced by a graft-versus-host reaction or by contact hypersensitivity. In the mouse, previous studies have suggested that Ia antigens in skin are restricted to epidermal Langerhans cells and it was thought that these were the targets for Ia-dependent rejection of skin allografts. The results presented here show that Ia antigens in mouse allografts are also present on the vascular endothelium but their expression is variable and dependent on the immunological status of the recipient. These findings suggest that vascular endothelial cells can act as targets in Ia-incompatible skin allograft rejection.

The role of complement in the induction of acute antibody-mediated vasculitis of rat skin grafts in the mouse.

Rat skin grafts carried by immunosuppressed mice can be acutely destroyed by intravenous administration of mouse anti-rat antibody. The velocity of the reaction and the histologic sequence of events depend on the amount of antibody administered: low doses give an Arthus-like rejection, whereas at high doses a Shwartzman-like pattern occurs. Depletion of C3 by cobra venom factor treatment did not prevent acute rejection after intravenous injection of high doses of antiserum but changed the reaction from a Shwartzman-like to an Arthus-like pattern. Conversely, supplementary administration of rabbit complement caused a violent Shwartzman-like graft destruction after injection of low doses of antibody, which in complement-normal mice gave an Arthus-like reaction. The results show that complement can greatly amplify the antibody-mediated immune vasculitis and can substantially modify its histologic pattern. It is, however, not an absolute requirement for the occurrence of the destructive process.

Patterns of vascular damage in the antibody-mediated rejection of skin xenografts in the mouse.

Established rat skin grafts carried by immunosuppressed mice were acutely destroyed by an intravenous administration of mouse antirat lymphocyte serum. The histologic pattern of destruction was dependent on the amount of antiserum administered. At low doses (0.01 ml) an Arthus-like reaction was seen with early accumulation of granulocytes. At high doses (0.25 ml) a Shwartzman-like pattern occurred, with early intravascular thrombosis and without evident participation of granulocytes in the initial phases. Groups of mice that received intermediate doses showed graft changes that were transitional between these two types of destruction. Similar histologic patterns have been described in clinical transplantation. Our results show that they are not fundamentally different and that the severity of the triggering reaction determines which of either type will occur.