Blockade of vascular adhesion protein-1 inhibits lymphocyte infiltration in rat liver allograft rejection - PubMed (original) (raw)

Blockade of vascular adhesion protein-1 inhibits lymphocyte infiltration in rat liver allograft rejection

Timi Martelius et al. Am J Pathol. 2004 Dec.

Abstract

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation, but its functional role in vivo has not been tested in any rodent model. Here we report the effects of VAP-1 blockade on rat liver allograft rejection. BN recipients of PVG liver allografts (known to develop acute rejection by day 7) were treated with 2 mg/kg anti-VAP-1 (a new anti-rat VAP-1 mAb 174-5) or isotype-matched irrelevant antibody (NS1) every other day (n = 6/group) and one group with anti-VAP-1 2 mg/kg daily (n = 7). On day 7, samples were collected for transplant aspiration cytology, histology, and immunohistochemistry. Lymphocyte infiltration to the graft was clearly affected by VAP-blockade. The total inflammation, mainly the number of active lymphoid cells, in transplant aspiration cytology was significantly decreased in animals treated with anti-VAP-1 (4.7 +/- 1.0 and 2.4 +/- 1.0 corrected increment units, respectively) compared to control (6.6 +/- 1.0) (P < 0.05). In histology, the intensity of portal inflammation was significantly decreased (P < 0.05). The amount of T cells expressing activation markers diminished. This is the first demonstration in any prolonged in vivo model that VAP-1 plays an important role in lymphocyte infiltration to sites of inflammation, and, in particular, liver allograft rejection.

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Figures

Figure 1

Figure 1

Characterization of an anti-rat VAP-1 mAb. A: Immunohistochemical staining of human peripheral lymph nodes with mAb 174–5 yields identical results with that of a prototype anti-VAP-1 mAb (both stain HEV). NS1 is a negative control mAb. 174–5 stains small vessels (white arrows) in the transverse sections of human and rat gut. L, lumen. Black arrows point to mast cells that are brown due to their endogenous peroxidase. B: FACS staining of Ax cells transfected with mock or VAP-1 plasmid with NS-1 negative control mAb and with 174–5. C: In HEV binding assay, mAb 174–5 inhibits significantly lymphocyte binding to HEV when compared to a negative control.

Figure 2

Figure 2

A: Expression of VAP-1 in normal human liver. Paraffin section, mAb TK10–79 as primary antibody. Original magnification, ×200. B: VAP-1 expression in normal rat liver. Frozen section, immunoperoxidase staining, mAb 174–5 as primary antibody. Original magnification, ×400. D: Rat liver allograft with acute rejection 7 days post-transplantation. Frozen section, immunoperoxidase staining, mAb 174–5 as primary antibody. Original magnification, ×400. C, D, and F: Intraluminal expression of VAP-1. Frozen sections, immunoperoxidase staining detecting the primary antibody given i.v. Original magnification, ×20. C: Normal liver, 30 minutes after i.v. injection of 2 mg/kg anti-VAP-1. E: Allografted liver, 7 days post-transplantation, 30 minutes after single i.v. injection of 2 mg/kg anti-VAP-1. Intraluminal antibody binding is inhibited. F: Allografted liver, 7 days post-transplantation, treated with daily injections of 2 mg/kg anti-VAP-1. There is clear intraluminal expression of VAP-1.

Figure 3

Figure 3

Effect of anti-VAP-1 treatment on the rejection response measured by transplant aspiration cytology on day 7 post-transplantation. The results are given in corrected increment units (CIU). In the box plots median, interquartile range (box) and adjacent values (whiskers) are shown. Outliers are shown as dots. Significant difference (P < 0.5) compared to control is indicated by asterisks.

Figure 4

Figure 4

Histopathological findings 7 days after liver transplantation. Control antibody- (A) and anti-VAP-1- treated (2 mg/kg/d) (B) liver allografts. Original magnification, ×100. C. Dot plots show the individual scores of the grafts. For control antibody group n = 6, for anti-VAP-1 2 mg/kg/2 days group n = 6, and for anti-VAP-1 2 mg/kg/d group n = 7. Significant difference compared to control is indicated by asterisk.

Figure 5

Figure 5

Expression of leukocyte surface markers CD3, CD4, CD8, IL-2 receptor, VLA-4, LFA-1, CD45RC, rat B-cell marker HY3004, and CD2 in the liver allografts. The results are expressed as percentage of positive label of the total surface area, except B cells as mean number of positive cells/field. In the box plots median, interquartile range (box) and adjacent values (whiskers) are shown. Outliers are shown as dots. Significant difference (P < 0.05) compared to control is indicated by asterisks.

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