Alternative macrophage activation-associated transcripts in T-cell-mediated rejection of mouse kidney allografts.

Macrophages display two activation states that are considered mutually exclusive: classical macrophage activation (CMA), inducible by IFNG, and alternative macrophage activation (AMA), inducible by IL4 and IL13. CMA is prominent in allograft rejection and AMA is associated with tissue remodeling after injury. We studied expression of AMA markers in mouse kidney allografts and in kidneys with acute tubular necrosis (ATN). In rejecting allografts, unlike interferon gamma (IFNG) effects and T-cell infiltration that developed rapidly and plateaued by day 7, AMA transcripts (Arg1, Mrc1, Mmp12 and Ear1) rose progressively as tubulitis and parenchymal deterioration developed at days 21 and 42, despite persistent IFNG effects. AMA in allografts was associated with transcripts for AMA inducers IL4, IL13 and inhibin A, but also occurred when hosts lacked IL4/IL13 receptors, suggesting a role for inhibin A. Kidneys with ATN injured by ischemia/reperfusion also had increased expression of AMA markers and inhibin A. Thus kidneys undergoing T-cell-mediated rejection progressively acquire macrophages with alternative activation phenotype despite strong local IFNG effects, independent of IL4 and IL13. Although the mechanisms and causal relationships remain to be determined, high AMA transcript levels in rejecting allografts are strongly associated with and may be a consequence of parenchymal deterioration similar to ATN.

The early course of kidney allograft rejection: defining the time when rejection begins.

We studied the early events in mouse kidney allografts and isografts to define when allorecognition begins and when alloimmune tissue injury begins. Allografts but not isografts showed T-cell infiltration in perivascular areas from day 1, but tubulitis and arteritis did not develop until day 7. Flow cytometry confirmed the early allospecific CD3(+)CD8(+) T-cell infiltrate. At day 1, both allografts and isografts showed extensive transcriptome changes, reflecting the response to surgery, but only allografts showed expression of interferon-gamma (IFN-gamma)-inducible transcripts and T-cell-associated transcripts. Although the number of CD68(+) myeloid cell numbers did not increase in day 1 isografts or allografts, mRNA expression for myeloid markers was increased in isografts and allografts, suggesting activation of resident cells of the macrophage-dendritic cell series (MMDCs) in response to injury, followed by increased CD68(+) cell numbers from day 2. By day 3, an interstitial T-cell and MMDC infiltrate was established in allografts, corresponding with the emergence of allospecific tissue injury, as reflected by decreased parenchymal transcripts. Thus, in renal allografts, allorecognition by T cells occurs in perivascular sites by day 1, but alloimmune parenchymal damage begins at day 3, coinciding with the emergence of the interstitial T-cell-MMDC infiltrate.

FOXP3 expression in human kidney transplant biopsies is associated with rejection and time post transplant but not with favorable outcomes.

Expression of the transcription factor forkhead box P3 (FOXP3) in transplant biopsies is of interest due to its role in a population of regulatory T cells. We analyzed FOXP3 mRNA expression using RT-PCR in 83 renal transplant biopsies for cause in relationship to histopathology, clinical findings and expression of pathogenesis-based transcript sets assessed by microarrays. FOXP3 mRNA was higher in rejection (T-cell and antibody-mediated) than nonrejection. Surprisingly, some native kidney controls also expressed FOXP3 mRNA. Immunostaining for FOXP3 was consistent with RT-PCR, showing interstitial FOXP3+ lymphocytes, even in some native kidney controls. FOXP3 expression correlated with interstitial inflammation, tubulitis, interstitial fibrosis, tubular atrophy, C4d positivity, longer time posttransplant, younger donors, class II panel reactive antibody >20% and transcript sets reflecting inflammation and injury, but unlike these features was time dependent. In multivariate analysis, higher FOXP3 mRNA was independently associated with rejection, T-cell-associated transcripts, younger donor age and longer time posttransplant. FOXP3 expression did not correlate with favorable graft outcomes, even when the analysis was restricted to biopsies with rejection. Thus FOXP3 mRNA expression is a time-dependent feature of inflammatory infiltrates in renal tissue. We hypothesize that time-dependent entry of FOXP3-positive cells represents a mechanism for stabilizing inflammatory sites.

Comparing microarray versus RT-PCR assessment of renal allograft biopsies: similar performance despite different dynamic ranges.

In renal allografts, assessing gene expression can add relevant diagnostic information to histopathology. Results can be expressed as single genes or gene sets, representing pathogenesis-based transcript sets (PBTs): cytotoxic T-cell-associated, interferon gamma- induced or decreased kidney parenchymal transcripts. Two technology platforms are available: RT-PCR and microarrays. We compared RT-PCR, U133plus2.0 microarrays and histopathology in 86 biopsies. We compared 13 potentially diagnostic genes as RT-PCR probes to microarray-derived PBTs, 'mini'-PBTs (small sets of 3-5 transcripts) and a histology classifier. Most RT-PCR probes (10/13) correlated well with the corresponding microarray probe sets (r > 0.8). Exceptions included FASLG and CD8B1 microarray probe sets, which were not performing on microarrays but were detectable by RT-PCR most likely due to differences in sensitivity. In general, RT-PCR showed greater dynamic range, detecting small changes in normal kidneys, but RT-PCR and microarrays gave similar results in abnormal kidneys. Individual transcripts or mini-PBTs assessed by either platform correlated well with one another, with microarray PBTs and the histology classifier. Thus, microarrays and RT-PCR assessments agree strongly with one another and histopathology in assessing transplant inflammation, particularly, when results are expressed as PBTs or mini-PBTs. The dynamic range of both platforms was sufficient to detect the relevant changes in rejection.

The transcriptome of human cytotoxic T cells: similarities and disparities among allostimulated CD4(+) CTL, CD8(+) CTL and NK cells.

Transcripts expressed in cytotoxic T lymphocytes (CTL) have mechanistic and diagnostic importance in transplantation. We used microarrays to select CTL-associated transcripts (CATs) expressed in human CD4(+) CTL, CD8(+) CTL and NK cells, excluding transcripts expressed in B cells, monocytes and kidney. This generated three transcript sets: CD4(+)-associated, CD8(+)-associated and NK-associated. Surprisingly, many CATs were expressed in effector memory cells e.g. granzyme B/GZMB, interferon-gamma/IFNG. Transcript expression was very similar between CD4(+) and CD8(+) CTL. There were no transcripts highly selective for CD4(+) CTL or CD8(+) CTL: for example, cytotoxic molecule transcripts (perforin, granzymes, granulysin) were shared between CD8(+) CTL and CD4(+) CTL although expression remained higher in CD8(+) CTL. Transcripts that differentiated between CD8(+) CTL and CD4(+) CTL were primarily those shared between CD8(+) CTL and NK cells (e.g. NK receptors KLRC1, KLRC3, KLRD1, KLRK1). No transcripts could differentiate CD4(+) CTL from CD8(+) CTL but NK cell-associated transcripts could differentiate NK cells from CTL. This study serves as a foundation for the interpretation of CATs in rejecting allografts and highlights the extensive sharing of CATs among CD4(+) CTL, CD8(+) CTL and effector memory T cells.

The transcriptome of human cytotoxic T cells: measuring the burden of CTL-associated transcripts in human kidney transplants.

Having defined CTL-associated transcripts (CATs) in CTL in vitro, we used microarrays to quantify the burden of CAT sets compared with individual transcripts in human renal transplant biopsies with T-cell mediated rejection (TCMR). CAT sets in TCMR resembled diluted CTL RNA, maintaining overall hierarchy of expression relative to CTL in vitro. NK selective sets were not detected in TCMR, indicating the CATs mainly reflect T cells. We selected 25 highly expressed CATs that diluted quantitatively in kidney RNA (QCATs) and remained detectable after 32-fold dilution. QCAT burden in 14 kidneys with TCMR was 3 to 15% of CTL RNA, correlating with infiltration. One biopsy diagnosed as TCMR only by endothelialitis had little interstitial infiltrate and lowest CAT burden. CAT sets were more consistent than individual CATs such as perforin or granzyme B, which showed heterogeneity. In luster and principal component analysis, QCATs grouped biopsies with TCMR together, in close relationship to in vitro CTL. Thus QCAT sets robustly measure the burden of CTL and effector memory T cells in biopsies as %CTL RNA, in a manner not achieved by measurement of individual transcripts.

IFN-gamma prevents early perforin-granzyme-mediated destruction of kidney allografts by inducing donor class I products in the kidney.

Interferon-gamma (Ifng) protects organ allografts: mouse kidney allografts lacking Ifng receptors rapidly fail with massive ischemic necrosis around days 5 to 7, reflecting microcirculation failure. We hypothesized that Ifng protects the graft by preventing perforin-granzyme-mediated cytotoxic damage to the microcirculation by inducing class Ia and/or Ib products. We transplanted kidney allografts lacking Ifng receptors into various knockout hosts. The necrosis/congestion phenotype did not require host B cells or IL-4 and IL-13 receptors, but required the T-cell alloresponse: it did not occur if the hosts were syngeneic or T-cell deficient. However, host perforin-granzyme mechanisms were required: no necrosis developed if hosts lacked either perforin or granzymes A and B. The ability of Ifng to protect the allograft required donor class I products: allografts lacking class I products due to Tap1 or beta2 microglobulin deficiency developed a similar necrosis-congestion phenotype at day 7 despite Ifng receptors being present. Thus when host cytotoxic T cells infiltrate organ allografts, Ifng prevents their perforin-granzyme mechanism from compromising the microcirculation by a mechanism requiring donor class Ia or Ib products. We propose that donor class Ia or Ib products are needed to trigger inhibitory receptors on effector T cells.

Changes in the transcriptome in allograft rejection: IFN-gamma-induced transcripts in mouse kidney allografts.

We used Affymetrix Microarrays to define interferon-gamma (IFN-gamma)-dependent, rejection-induced transcripts (GRITs) in mouse kidney allografts. The algorithm included inducibility by recombinant IFN-gamma in kidneys of three normal mouse strains, increase in kidney allografts in three strain combinations and less induction in IFN-gamma-deficient allografts. We identified 40 transcripts, which were highly IFN-gamma inducible (e.g. Cxcl9, ubiquitin D, MHC), and 168 less sensitive to IFN-gamma in normal kidney. In allografts, expression of GRITs was intense and consistent at all time points (day 3 through 42). These transcripts were partially dependent on donor IFN-gamma receptors (IFN-gammars): receptor-deficient allografts manifested up to 76% less expression, but some transcripts were highly dependent (ubiquitin D) and others relatively independent (Cxcl9). Kidneys of hosts rejecting allografts showed expression similar to that observed with IFN-gamma injections. Many GRITs showed transient IFN-gamma-dependent increase in isografts, peaking at day 4-5. GRITs were increased in heart allografts, indicating them as generalized feature of alloresponse. Thus, expression of rejection-induced transcripts is robust and consistent in allografts, reflecting the IFN-gamma produced by the alloresponse locally and systemically, acting via host and donor IFN-gammar, as well as local IFN-gamma production induced by post-operative stress.

Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications.

A comprehensive survey of the extensive literature relevant to the evolution, physiology, biochemistry, regulation, and genetic engineering applications of plant aromatic L-amino acid decarboxylases (AADCs) is presented. AADCs catalyze the pyridoxal-5'-phosphate (PLP)-dependent decarboxylation of select aromatic L-amino acids in plants, mammals, and insects. Two plant AADCs, L-tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC), have attracted considerable attention because of their role in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids and benzylisoquinoline alkaloids, respectively. Although plant and animal AADCs share extensive amino acid homology, the enzymes display striking differences in their substrate specificities. AADCs from mammals and insects accept a broad range of aromatic L-amino acids, whereas TDC and TYDC from plants exhibit exclusive substrate specificity for L-amino acids with either indole or phenol side chains, but not both. Recent biochemical and kinetic studies on animal AADCs support basic features of the classic AADC reaction mechanism. The catalytic mechanism involves the formation of a Schiff base between PLP and an invariable lysine residue, followed by a transaldimination reaction with an aromatic L-amino acid substrate. Both TDC and TYDC are primarily regulated at the transcriptional level by developmental and environmental factors. However, the putative post-translational regulation of TDC via the ubiquitin pathway, by an ATP-dependent proteolytic process, has also been suggested. Isolated TDC and TYDC genes have been used to genetically alter the regulation of secondary metabolic pathways derived from aromatic amino acids in several plant species. The metabolic modifications include increased serotonin levels, reduced indole glucosinolate levels, redirected shikimate metabolism, increased indole alkaloid levels, and increased cell wall-bound tyramine levels.