While microvascular loss is strongly associated with chronic rejection 2-5, 12, 30, 31
, little is known about how inflammatory mediators specifically affect vascular flow during this process. The ability to attribute physiologic derangements to individual immune components requires an experimental model that can be interrogated for microvascular injury-associated ischemia. By focusing on the functionality of graft microvasculature, the definition of allograft health broadens within this study. (Online Table 1
summarizes experimental results, and Online Table 2
interprets these findings.) The cumulative data demonstrate that CD4+
T cells and complement independently mediate graft ischemia during acute rejection.
In the current study, CD4+
T cells were found sufficient, independent of antibody and complement, to induce microvascular injury-associated ischemia whereas CD8+
T cells, in isolation, did not induce microvascular injury-associated ischemia. Surprisingly, CD4 T cell-mediated injury did not recover in the absence of CD8+
T cells; CD8+
T cells were required for airway neovascularization to occur following CD4-mediated rejection. T cells have previously been implicated as important pro-angiogenic cells 32, 33
T cells contribute to the early phase of collateral vessel development in hind limb ischemia by promoting the recruitment of angiogenic CD4+
T cells in an IL-16-dependent manner 34
. CD8-recruitment of other angiogenic monocytes and macrophages, that are also key to vascular collateralization following ischemic injury, may also explain the requirement of CD8+
T cells to revascularize rejected airway transplants 34-36
Airway ischemia during acute rejection was closely associated with the presence of subepithelial fibrosis. Of note, in the absence of CD8+
T cells following acute rejection, the microvascular injury-associated ischemia is most prolonged, but subepithelial fibrosis is surprisingly limited, and collagen transcription, minimal. It is possible that the complete absence of perfusion to the transplant for many days also decreased the delivery of fibrosis-causing elements in the blood such as cytokines and fibrocytes 37
. The duration of airway ischemia closely correlated with the appearance of the overlying epithelium with a brief cessation of airway perfusion during rejection still allowing the preservation of an intact overlying columnar lining but prolonged ischemia being tightly associated with a flattened dysplastic-appearing overlying epithelium. Thus, as our other studies have recently demonstrated 3, 12
, microvascular injury-associated ischemia in airways, during acute rejection episodes, appear to be causally-linked to the airway remodeling observed in chronic rejection.
The following sequence of key results subsequently led to the discovery of two independent effector pathways for microvascular injury-associated ischemia: 1) Allografts in unreconstituted complement-replete RAG1−/− mice do not become ischemic. 2) CD4-reconstitution of RAG1−/− recipients results in transplant ischemia. 3) CD4-replete/C3-inhibited recipients demonstrate graft ischemia. 4) Adoptive transfer of donor-specific MHC Class II antibodies restores graft ischemia in B and T cell-deficient/complement-replete RAG1−/− recipients. 5) C3-deficient/antibody-replete/CD4-depleted WT recipients do not develop ischemia. The latter two results indicate that in the absence of CD4 cells, C3 and antibody must both be present for graft ischemia to occur. The cumulative results indicate that CD4+ T cells and antibody-dependent complement activation independently mediate microvascular injury-associated ischemia. Because C3-deficient/CD4-depleted WT recipients do not become ischemic, additional effector pathways are not required to explain the loss of perfusion in airway transplants.
Complement activation causes dysregulation of angiogenic factors required for normal fetal development, and C3 inhibition with Crry-Ig blocks the pathological increase in soluble VEGFR-1, a potent inhibitor of VEGF activity, and rescues pregnancies in mice 27
. CR2-Crry treatment up-regulated proangiogenic factors within the transplant, which may have caused the rapid reinvestment of microvessels into these rejected airways. C3−/−
recipients had a similar (although not identical) profile of upregulated proangiogenic factors associated with a faster revascularization period. A recent study in an animal model of retinopathy of prematurity mirrors these findings 38
. The loss of donor-derived microvasculature was evident in C3−/−
recipients; their rapid replacement with non-transgenic vessels is consistent with recipient vessels growing into the transplant as an accelerated response to increased graft-derived proangiogenic factors. C3−/−
recipients exhibited leakier and more dilated allograft vessels earlier in rejection for unknown reasons. In C3−/−
mice, thrombin substitutes for C3-dependent C5 convertase leading to the generation of C5a 39
; the latter complement component being an anaphylatoxin that can trigger vasodilation and increase capillary permeability. Given that the generation of thrombin is a feature of allograft rejection 40
, it is possible that excessive C5a activation could account for increased microvascular dilation and permeability in low/inhibited C3 conditions. Regardless of this finding, the ischemic burden of C3−/−
and CR2-Crry-treated animals was substantially less than in WT-rejecting animals. We have recently reported that manipulating airway allografts by selectively increasing proangiogenic factors by HIF-1α modulation similarly accelerates recipient-derived angiogenesis and limits fibrotic graft remodeling 12
This study strongly suggests that the duration of hypoxia and ischemia in allograft rejection is relevant to the subsequent development of significant airway remodeling as manifested by a loss of normal epithelium and increased subepithelial fibrosis. Lung transplant recipients are particularly vulnerable to the deleterious effects of airway hypoxia and ischemia. The lung is unique among solid organ transplants because of the lack of the surgical restoration of a vascular connection to the systemic circulation. Blood supply to the airways in lung transplant recipients, in contrast to the normal dual circulation, presumably comes from the deoxygenated pulmonary artery circulation 41
. Therefore, from the outset, lung transplant airways have an impaired microcirculation due to the lack of bronchial artery restoration, which, we have recently demonstrated, results in relative airway tissue hypoxia in lung transplant patients 42
. We have hypothesized that this baseline airway hypoxia may be a diathesis for chronic rejection in lung transplant recipients 42
, and therapies which preserve microvascular integrity may be especially relevant.
Profound tissue ischemia, undetectable by histology, may ‘silently’ occur during acute rejection in transplant recipients. Knowledge of the immune factors which cause microvascular-injury associated ischemia should help to more logically target therapeutics designed to preserve microvessel integrity. Captured perfusion and oxygenation data from this study reveal, paradoxically, that CD8+ T cell depletion interferes with angiogenesis in ischemic tissue and may harm graft recovery in patients. Further, this new physiologic information suggests that targeted complement inhibitors could safely synergize with conventional therapy during rejection episodes to prevent microvascular injury-associated ischemia and potentially limit the development of chronic rejection.