Robert L. Fairchild

Interferon action and apoptosis are defective in mice devoid of 2′,5′-oligoadenylate-dependent RNase L

2′,5′‐Oligoadenylate‐dependent RNase L functions in the interferon‐inducible, RNA decay pathway known as the 2‐5A system. To determine the physiological roles of the 2‐5A system, mice were generated with a targeted disruption of the RNase L gene. The antiviral effect of interferon α was impaired in RNase L−/− mice providing the first evidence that the 2‐5A system functions as an antiviral pathway in animals. In addition, remarkably enlarged thymuses in the RNase L−/− mice resulted from a suppression of apoptosis. There was a 2‐fold decrease in apoptosis in vivo in the thymuses and spleens of RNase L−/− mice. Furthermore, apoptosis was substantially suppressed in RNase L−/− thymocytes and fibroblasts treated with different apoptotic agents. These results suggest that both interferon action and apoptosis can be controlled at the level of RNA stability by RNase L. Another implication is that the 2‐5A system is likely to contribute to the antiviral activity of interferon by inducing apoptosis of infected cells.

TLR Engagement Prevents Transplantation Tolerance

In many experimental models, heart, pancreas and kidney allografts are accepted long‐term following costimulation‐targeting therapies, whereas skin, lung and intestine resist the induction of tolerance under the same regimens. We noted that a common feature of the resistant organs is their constant exposure to commensal microbes and hypothesized that these microorganisms may stimulate Toll‐like receptors (TLRs), promote alloresponses and prevent tolerance induction. This hypothesis prompts the predictions that TLR engagement at the time of transplantation should avert tolerance to heart allografts in animals treated with costimulation‐targeting therapies, whereas inhibition of TLR signaling should promote tolerance to skin allografts under the same conditions. Indeed, engagement of a single TLR was sufficient to prevent anti‐CD154‐mediated long‐term cardiac allograft acceptance and correlated with abolished intragraft recruitment of CD4+/FoxP3+ regulatory T cells and the development of linked‐suppression. Conversely, a lack of donor and recipient MyD88‐dependent signaling led to successful skin allograft acceptance in anti‐CD154‐treated animals. Thus, the status of TLR signaling contributes to the resistance versus susceptibility of organs to transplantation tolerance.

Neutralization of Groα and Macrophage Inflammatory Protein-2 Attenuates Renal Ischemia/Reperfusion Injury

Previous studies have provided strong evidence for a role for neutrophils in mediating pathology during reperfusion of ischemic tissues. CXC chemokines including interleukin-8, KC/Groα, and macrophage inflammatory protein (MIP)-2, direct neutrophils to tissue sites of inflammation. In the current study we tested the efficacy of antibodies to KC/Groα and MIP-2 in inhibiting neutrophil infiltration into kidneys during reperfusion after 1 hour of warm ischemia using a mouse model. KC mRNA and protein were produced within 3 hours after reperfusion of the ischemic kidneys. MIP-2 mRNA and protein were twofold to fourfold lower than KC and were at low levels until 9 hours after reperfusion. Only 60% of mice subjected to ischemia/reperfusion injury survived to day 3 after reperfusion. Treatment with rabbit neutralizing antibodies to both KC and MIP-2 inhibited neutrophil infiltration into ischemic kidneys during reperfusion, restored renal function as assessed by decreased serum creatinine and urea nitrogen levels to near normal levels, and resulted in complete survival of treated animals. Finally, treatment with both antibodies significantly reduced histologically graded pathology of kidneys subjected to ischemia/reperfusion injury. Collectively, the results indicate the efficacy of neutralizing the chemokines directing neutrophils into ischemic kidneys during reperfusion to inhibit this infiltration and attenuate the resulting pathology.

Urinary-Cell mRNA Profile and Acute Cellular Rejection in Kidney Allografts

BACKGROUND The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. METHODS We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. RESULTS A three-gene signature of 18S ribosomal (rRNA)-normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operating-characteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer-Lemeshow test indicated good fit (P=0.77). In an external-validation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P=0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti-interleukin-2 receptor antibodies from those who received T-cell-depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P=0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). CONCLUSIONS A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts. (Funded by the National Institutes of Health and others.).

Monokine Induced by IFN-γ Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-γ inducible protein 10 (IP-10) and monokine induced by IFN-γ (Mig) in acute rejection of A/J (H-2a) cardiac grafts by C57BL/6 (H-2b) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7–9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17–19), intra-allograft expression of macrophage-inflammatory protein-1α, -1β, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.

Early Chemokine Cascades in Murine Cardiac Grafts Regulate T Cell Recruitment and Progression of Acute Allograft Rejection

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2a) and syngeneic heart grafts in C57BL/6 (H-2b) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene α (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1β and 1α. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-γ-inducible protein-10 and monokine induced by IFN-γ, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.

Chemokines: directing leukocyte infiltration into allografts

Chemokines have been shown to play a critical role in the recruitment of leukocytes to transplanted organs. Animal models and clinical studies have demonstrated predictable temporal and spatial correlations between chemokine production and leukocyte infiltration into allografts. Antagonism of chemokines or chemokine receptors has been shown to delay leukocyte infiltration and prolong graft function, demonstrating an important role for chemokines in allograft rejection.

Chemokine and chemokine receptor gene expression indicates acute rejection of human cardiac transplants.

Background. Factors directing T-cell infiltration into allografts during acute rejection remain poorly defined. Chemokines have been shown to mediate leukocyte recruitment into allografts in animal models of rejection. The goal of this study was to test the presence and levels of chemokine and receptor gene expression in serial endomyocardial biopsy specimens from heart transplant patients and to correlate the levels observed with histopathologic rejection grade. Methods. Three hundred sixteen serial endomyocardial biopsy specimens from 30 heart transplant patients were obtained during the clinically scheduled surveillance heart biopsy program. The follow-up period was 1 year. The expression of interferon (IFN)-&ggr; inducible protein (IP)-10, monokine induced by IFN-&ggr; (Mig), interferon-inducible T-cell alpha chemoattractant (I-TAC), regulated on activation normal T-cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, interleukin (IL)-8, and the receptors CXCR3 and CCR5 were tested using quantitative, real-time polymerase chain reaction. Biopsy samples were examined histologically to assign rejection grade. Results. Expression of IP-10, Mig, I-TAC, RANTES, CXCR3, and CCR5, but not MCP-1 and IL-8, increased significantly in both grade 2 and grade 3 rejection (P ≤0.0096). These increases returned to normal after treatment with pulse steroid therapy to treat the rejection episode. Conclusion. The expression of IP-10, Mig, I-TAC, RANTES, CXCR3, and CCR5 in cardiac allografts significantly correlates with the presence and grade of acute rejection.

Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.

Multicenter Validation of Urinary CXCL9 as a Risk‐Stratifying Biomarker for Kidney Transplant Injury

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision‐making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation‐01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy‐proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6‐month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5–99.3% NPV). Our results support using urinary CXCL9 for clinical decision‐making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.