Increased expression of peripheral blood leukocyte genes implicate CD14+ tissue macrophages in cellular intestine allograft rejection

Abstract

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.

Figure 1

Study design. Twenty-nine children were evaluated in the present study, 20 in the test cohort and 9 in an independent validation cohort. The test cohort of 20 children provided i) late post-ITx blood samples for candidate gene discovery with microarrays, ii) pre-ITx and early post-ITx blood samples for quantitative real-time PCR replication of candidate genes, iii) random blood sample for cellular localization of the replicated candidate gene, and iv) allograft biopsy tissue to identify the candidate cell associated with rejection along with its time-dependent changes in postperfusion and late post-ITx allograft biopsies. Postperfusion biopsies are obtained within 4 hours of allograft implantation, and within 24 hours of pre-ITx blood sampling. All other biopsies correspond to blood samples used for gene expression studies. An independent validation cohort of nine children provided additional biopsy tissue to confirm the time course of the candidate cell associated with ITx. NR, nonrejector; R, rejector.

Figure 2

A: Quantitative real-time PCR replication as fold-change increase among rejectors, compared with nonrejectors, for eight candidate genes at three different time periods with respect to transplantation in the test cohort. Gene expression findings are also replicated late after ITx in an independent cohort of nine children (four rejectors, five nonrejectors). B: Summary data for frequencies of peripheral blood CD14⁺ monocytes, CD16⁺/56⁺ NK cells, and CD8⁺ Tc (all from four nonrejectors) that express CCL5, and a comparison of CCL5⁺CD14⁺ monocytes between five rejectors and four nonrejectors. C: Scatterplots illustrate differences in CCL5-expressing subsets from one rejector and one nonrejector.

Figure 3

A: Canonical pathways (arrows), antigen-presentation as a top-ranked biological function (enclosed within oval), and inflammatory disease as one of the top-ranked disorders (enclosed within triangle) suggested by different combinations of candidate genes in Ingenuity Systems pathway analysis. B: Significantly higher intragraft CD14⁺ MDM counts per high-power field in allograft biopsies from rejectors, compared with rejection-free biopsies from nonrejectors. No statistical differences were seen for CD163⁺ macrophages and CCL5-stained cells per high-power field.

Figure 4

Sequential changes in intragraft CD14⁺ MDM per high-power field (A) and the immunoreactivity index of allospecific CD154⁺ TcM (B) in peripheral blood in rejectors and nonrejectors from the test cohort. C: Changes in intragraft CD14⁺ MDM per high-power field in serial allograft biopsies from the validation cohort. D: Composite of serial hematoxylin-eosin stained biopsies with peroxidase stained CD14⁺ MDM (dark brown) from one nonrejector and one rejector. Magnification is 40×. ACR, acute cellular rejection; hpf, high-power field.