Role of Fibrinogen-Like Protein 2 Prothrombinase/Fibroleukin in Experimental and Human Allograft Rejection1
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免疫学杂志 2005年第11期
Abstract
Immune coagulation is a major contributor to the pathogenesis of xenograft rejection, viral-induced hepatocellular injury and cytokine-induced fetal loss syndrome. In this study, we investigated the contribution of the novel gene product, fibrinogen-like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection. Using a mouse heterotopic cardiac transplant model, mouse fgl2(mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4- and CD8-positive T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Although mfgl2-deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2-deficient mice were prolonged and deposition of intravascular fibrin was diminished. Treatment of wild-type mice with a neutralizing anti-fgl2 Ab ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival. To address further the relevance of fgl2 in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. Human fgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8+ T cells, mature B cells (plasma cells), and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with human fgl2 expression. These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection.
Introduction
Fibrinogen-like protein 2 (fgl2)4/fibroleukin, also known as fgl2 prothrombinase, has recently been cloned and identified and shown to belong to the fibrinogen family of proteins. Mouse fgl2 (mfgl2) and human fgl2 (hfgl2) have been localized to chromosomes 5 and 7, respectively (1, 2, 3). fgl2 prothrombinase has been shown previously to have the attributes of a serine protease capable of directly cleaving prothrombin to thrombin leading to fibrin deposition (2). Several studies indicate that mfgl2 is involved in experimental xenograft rejection by mediating "immune coagulation," fibrin deposition, and microthrombus formation, leading to classical pathological changes of acute vascular rejection (4). The role of fgl2 in allorejection has not been examined in detail. Previously, Hancock et al. (5) have suggested that mfgl2 expression is correlated with allograft rejection, and strategies known to prevent allorejection including infusion of anti-CD154 prevent expression of mfgl2. However, this group recently also suggested that disruption of mfgl2 did not alter type 1 immunity or fibrin deposition associated with allograft rejection (6).
In this present study, we investigated the expression of fgl2 in acute allograft rejection, first using a mouse heterotopic cardiac transplant model, and subsequently extending our studies to examine hfgl2 expression in rejecting human renal allografts. Expression of fgl2 was studied by both in situ hybridization and immunochemistry. mfgl2 transcripts were highly expressed in macrophages, T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Treatment of mice with a high-titered neutralizing anti-fgl2 polyclonal Ab ameliorated the pathological injury and resulted in measurably increased graft survival. In rejecting human renal allografts, hfgl2 mRNA transcripts and protein expression correlated with the presence of rejection. Collectively, these studies suggest that fgl2 expression may be critical to the pathogenesis of both experimental and human allograft rejection and provide a rationale for targeting the fgl2 gene in an attempt to modulate allograft rejection.
Materials and Methods
Statistical analysis
Quantitative data were expressed as mean ± SD. Statistical analysis was conducted by using independent t test to compare effects of treatment on mean grade of rejection grade. Survival data were measured using a Kaplan-Meier model, and overall strata comparisons were made using log rank (Mantel-Cox) tests. The analysis was conducted on the statistical program for social sciences (SPSS), version 13.0, for Windows. A value of p < 0.05 was considered statistically significant.
Results
mfgl2 expression in rejecting grafts post-cervical heterotopic cardiac allotransplantation in mice
Hearts recovered from mice that underwent cervical heterotopic cardiac isogeneic or allogeneic transplantation at predetermined times posttransplantation (days 1, 3, 5, and 7) were examined for the presence of rejection (Table IV; Fig. 1, A–D) and expression of mfgl2. Transplantation of isogeneic grafts was used as controls. Expression of mfgl2 was examined at both the mRNA and protein levels by in situ hybridization and immunohistochemistry, respectively. Expression of mfgl2 was first detected at day 1 posttransplant, and expression of mfgl2 increased on days 3, 5, and 7 (Fig. 1, E–H and J–M). mfgl2 was seen primarily in infiltrating mononuclear cells and endothelial cells of the microvasculature, which correlated with the severity of the histopathological findings in rejecting grafts. There was little or no mfgl2 expression in grafts from the isogeneic group where there were no histological signs of rejection (Fig. 1, I–N).
Cellular source of mfgl2 and fibrin deposition in rejecting grafts in mice
By serial section staining, the majority of CD68+, CD4+, and CD8+ cells showed high expression of mfgl2 protein in rejecting grafts (Fig. 2, A–F). Fibrin deposition was also detected in the endothelium of microvascular vessels by immunoperoxidase staining by day 5 (Fig. 2, G and H).
Effects of fgl2 polyclonal Ab on the histopathological improvement in rejecting grafts post-cervical heterotopic cardiac allotransplantation in mice
The pathological grades of the rejecting cardiac grafts from mice treated with or without fgl2 polyclonal Ab based on a modification of the Banff criteria proposed by Billingham et al. (9) (Table II) are summarized in Table IV and Fig. 3. Grade of rejection of fgl2 Ab-treated grafts was statistically reduced when compared with untreated or control Ab-treated allografts on days 3, 5, and 7, whereas control Ab treatment had no effect on rejection grade (Fig. 3B). The histological changes within the rejecting cardiac grafts were improved post-fgl2 polyclonal treatment as shown in Fig. 3A, A–C, compared with that in Fig. 1, B–D, or Fig. 3AD, in which no Ab treatment or a control Ab was used.
Effect of fgl2 polyclonal Ab on survival of rejecting cardiac grafts post-cervical heterotopic heart allotransplantation in mice
Grafts from mice that had received two injections of 500 μg of fgl2 neutralizing polyclonal Ab had a modest, but statistically significant increase in survival compared with grafts from untreated mice or mice injected with a control polyclonal Ab by log rank (Mantel-Cox) analysis followed by a paired t test for individual groups. The mean survival time of Ab-treated mice was with a mean survival time of 9.4 ± 1.1 days compared with a survival time of 7.3 ± 0.5 days of untreated mice; and 7.3 ± 0.6 days of mice receiving control Ab (Fig. 4). Additionally, mice receiving two injections of Fab' equivalent to 500 μg of polyclonal anti-fgl2 (group E) were protected to the same degree as mice receiving the native anti-fgl2 molecule, with a mean survival of 9.7 + 1.3 days. These data are consistent with the hypothesis that protection occurs through neutralization of fgl2 rather than the involvement of other cells or molecules.
Heart grafts from BALB/cJ mice transplanted into mfgl2-deficient mice were universally rejected on day 6.4 ± 1.2, similar to transplantation of heart grafts from BALB/cJ mice, which were transplanted into fgl2+/+ littermate controls. In contrast, heart grafts from fgl2-deficient mice transplanted into BALB/cJ mice had a prolonged survival but were ultimately rejected on day 12.2 ± 1.4 (Table V).
Immunohistochemical assessment of hfgl2 expression in patients with renal acute allograft rejection
To address the relevance of hfgl2 in human acute allograft rejection, we studied patients with renal acute allograft rejection (Table III). Twenty-one of the patients were male, and 10 were female. The mean time to first rejection posttransplant was 14.9 + 24.1 mo. Grafted renal tissues with histopathological grades IB, IIA, IIB, and III were strongly and uniformly positive for hfgl2 expression, whereas IA lesions had less intense hfgl2 positivity, indicating a close association of hfgl2 expression with the histopathologic rejection and fibrin deposition. Expression of hfgl2 was seen primarily in renal tubule cells, in infiltrating mononuclear cells, and in the endothelium of small renal blood vessels and glomerular capillary wall in close proximity to fibrin deposits (Fig. 5).
By dual-staining immunohistochemistry, CD68+ (macrophages) and CD8+ (T cells) were shown to be the cellular source of hfgl2 (Fig. 6, A–D, respectively). Fibrin deposition was seen within the microvascular vessels, glomerular capillary wall and matrix, and the surface of infiltrating mononuclear cells in the blood vessels by immunoperoxidase staining in association with hfgl2 expression (Fig. 6, E and F).
Discussion
Fibrin deposition is a common element of many types of immunological reactions including delayed-type hypersensitivity, autoimmune disease, graft rejection, and the response to infectious agents (11, 12). Induction of procoagulant molecules on leukocytes and endothelial cells is thought to play an important role in the pathogenesis of thrombin generation and fibrin deposition at the sites of inflammation. This has been observed in the case of tissue factor, through the widespread constitutive expression of this molecule in subendothelial and perivascular tissues, suggesting that its major function is in initiating coagulation to achieve hemostasis after endothelial cell disruption.
Procoagulants other than tissue factor, which are induced specifically by immune mediators, may also play a critical role in regulating localized fibrin deposition. fgl2, also known as fibroleukin, is a 70-kDa type-2 transmembrane protein, which has been postulated to directly convert prothrombin to thrombin in the absence of factor VII or factor X (13). fgl2 has been shown previously to play a critical role in the pathogenesis of fulminant hepatitis induced by murine hepatitis virus strain 3 (MHV-3) (1, 7). Murine, porcine, and human fgl2 have now been cloned and sequenced, and fgl2 encodes the mouse fibrinogen-like protein (musfiblp), a previously described gene isolated from CTLs that shares significant homology to fibrinogen - and -chains (14, 15, 16). The murine and human proteins share 78% overall identity, with greater conservation at the C terminus. The C terminus of fgl2 corresponds to a highly conserved region that is found in fibrinogen as well as in other functionally unrelated fibrinogen-related proteins such as tenascin, ficolin, and angiopoietin (17, 18, 19).
In the context of innate immune activation, induction of fgl2 in macrophages and endothelial cells has been shown to contribute to the pathogenesis of tissue factor-independent fibrin deposition and organ injury. In support of its role as a coagulant are the observation that neutralizing Abs to mfgl2 prevent both fibrin deposition and death from MHV-3 infection (20). Recent studies have also shown that inhibition of reticuloendothelial cell mfgl2 expression through the use of gene-targeted fgl2-deficient (fgl2–/–) mice results in the prevention of MHV-3-induced fibrin deposition, liver injury, and death (7). Furthermore, we have recently reported that murine and human fgl2 prothrombinase/fibroleukin are highly expressed in endothelium, macrophages, and lymphocytes in xenograft rejection (4).
In the current study, elevated expression of mfg12 mRNA and protein was observed on vascular endothelial cells and infiltrating leukocytes, including macrophages, CD4- and CD8-positive T lymphocytes, in rejecting cardiac allografts in a mouse heterotopic cardiac transplant model, in association with deposits of fibrin. Treatment of mice with a high-titered neutralizing anti-mfgl2 polyclonal Ab reduced the pathological injury and led to modest but statistically significant increase in graft survival. The fact that Fab' anti-fgl2 was as protective as the native Ab suggests that the protection observed follows from neutralization of mfgl2 and is independent of other (FcR+) cells/molecules, including complement, in the environment. Furthermore, transplanted hearts from mfgl2–/– mice were largely devoid of fibrin deposition resulting in prolonged graft survival, whereas grafts from wild-type mice transplanted into mfgl2–/– recipients were rejected in a similar tempo, and with similar histopathology, as fgl2+/+ littermates. Collectively, these data provide evidence for a role for endothelial cell fgl2 expression in the fibrin deposition associated with allotransplant rejection and suggest that strategies designed to prevent endothelial cell fgl2 expression may prove of benefit to improving allotransplant graft survival. The data are further supported from patient studies in which increased fgl2 and fibrin expression were found mainly in renal tubule cells, infiltrating macrophages, CD8+ T cells, and plasmacytes, as well as endothelial cells in rejecting renal grafts.
Recent studies from Hancock et al. (6) have also examined the relevance of mfgl2 to fibrin deposition in allorejection. Their studies, similar to ours, showed that mfgl2–/– recipient mice rejected donor heart allografts in a fashion analogous to wild-type mice. Collectively, these studies support the hypothesis that (donor) endothelial cell production of mfgl2, rather than an immune-activated infiltrating leukocyte population, accounts for the fibrin deposition in allorejection (6). These data are further supported by our recent studies in xenotransplantation in which fibrin deposition associated with xenograft rejection was largely intravascular rather than associated with infiltrating inflammatory cells (21). Furthermore, xenografts from mfgl2–/– mice transplanted into rats were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium accounts for fibrin deposition of allo- and xenotransplant rejection.
It is important to appreciate that fgl2 along with other members of the fibrinogen family of molecules have also been implicated in delivery of signals, which activate various arms of the innate immune system (22). Accordingly, some of the effects mediated by alteration of fgl2 expression might reflect not simply altered thrombin deposition, but also an altered immune activation. As but one example, Chan et al. (23) reported that fgl2 modified expression of costimulatory molecules on developing dendritic cells, with a profound "downstream" effect on cytokine induction following allostimulation. Current studies from our laboratory support the hypothesis that an additional immunomodulatory role for fgl2 does indeed follow binding to a receptor expressed in multiple tissues (R. Liu, manuscript in preparation).
Although not the focus of this paper, the molecular pathways for induction of fgl2 have recently been studied. In murine hepatitis virus infection, nucleocapsid protein induces transcription of fgl2 through the transcription factor hepatic nuclear factor 4 and its cognate receptor (24, 25). In transplantation, fgl2 transcription appears to be regulated by cytokines. Macrophage induction of fgl2 is induced by IFN-, whereas preliminary data suggest that fgl2 transcription in endothelial cells occurs in response to TNF- but not IFN- (6). In contrast, induction of tissue factor is NF-B dependent, and, unlike fgl2, IFN- inhibits tissue factor transcription (M. F. Liu, unpublished data).
The importance of fgl2 to allograft rejection is supported first by the observation that expression of fgl2 correlates with rejection (6) and also by the fact that administration of neutralizing Ab to fgl2 diminished the pathological injury and improved graft survival in a similar fashion to that previously reported in murine hepatitis infection (20).
In the development of late interstitial fibrosis seen in cases of chronic rejection, a great deal of attention has been paid to the early fibrin deposition in the matrix in the rejected grafts. Acute rejection is known to be one of the high risk factors for this phenomenon (26, 27). An important observation in this current study in both the experimental mouse model and in humans is the finding of high expression of fgl2 prothrombinase in infiltrating cells within the matrix or interstitial region of the kidney. Interestingly, the expression of fgl2 was in seen in close association with local fibrin deposition, consistent with a role for fgl2 in the pathogenesis of chronic allograft rejection, although further studies are necessary to define a role for fgl2 in chronic rejection.
In summary, these studies in both a murine model and in human renal allograft rejection provide evidence suggestive of a role for fgl2 in the thrombosis associated with acute allograft rejection. The data indicate that endothelial cells rather than leukocyte fgl2 expression accounts for intravascular fibrin deposition. Although not part of this study, data generated by our group and that of Hancock et al. suggest that one mechanism of altered fgl2 transcription is through elaboration of cytokines including IFN- and TNF, although additional studies are necessary to determine whether other cytokines are involved in the induction of fgl2. The elaboration of fgl2 during acute allograft rejection and the interstitial fibrin deposition may lead to further injury and development of chronic allograft rejection, but additional studies are required to firmly establish the role of fgl2 in this process, and in particular a potential immunomodulatory role for fgl2 in this process. These studies provide a rationale to target fgl2 for therapeutic intervention in an attempt to ameliorate both acute and chronic allograft rejection.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Science Fund for Distinguished Young Investigators (30225040 (to Q.N.), 30123019 (to X.L.)) from the Natural Science Foundation of China (NSFC), NSFC Operating Fund 30100171 and 30170846, National Key Basic Research Program of China (2001CB510008), and the Canadian Institutes for Health Research Grant FRN33780
2 Address correspondence and reprint requests to Dr. Qin Ning, Laboratory of Infectious Immunology and Department of Infectious Disease, Tongji Hospital, 1095 Jie Fang Avenue, Wuhan 430030, China. E-mail address: qning{at}tjh.tjmu.edu.cn
3 Current address: Department of Immunology, Medical College of Chinese People’s Armed Police Force, Tianjin 300162, China.
4 Abbreviations used in this paper: fgl2, fibrinogen-like protein 2; mfgl2, mouse fgl2; hfgl2, human fgl2; Dig, digoxigenin; AP, alkaline phosphatase; MHV-3, murine hepatitis virus strain 3.
Received for publication August 31, 2004. Accepted for publication March 4, 2005.
References
Ding, J. W., Q. Ning, M. F. Liu, A. Lai, K. Peltekian, L. Fung, C. Holloway, H. Yeger, M. J. Phillips, G. A. Levy. 1998. Expression of the fgl2 and its protein product (prothrombinase) in tissues during murine hepatitis virus strain-3 (MHV-3) infection. Adv. Exp. Med. Biol. 440: 609-618.
Levy, G. A., M. Liu, J. Ding, S. Yuwaraj, J. Leibowitz, P. A. Marsden, Q. Ning, A. Kovalinka, M. J. Phillips. 2000. Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis. Am. J. Pathol. 156: 1217-1225.
Yuwaraj, S., J. Ding, M. Liu, P. A. Marsden, G. A. Levy. 2001. Genomic characterization, localization, and functional expression of FGL2, the human gene encoding fibroleukin: a novel human procoagulant. Genomics 71: 330-338.
Levy, G. A., J. W. Ding, D. Weiner, Q. Ning, L. Fung, A. Marinov, R. Gorczynski, M. J. Philips. 1999. The role of fibrinogen-like protein (fgl2/fibroleukin) in xenograft rejection: induction of fgl2 prothrombinase by xenoserum. Hepatology 30: 109.
Hancock, W. W., M. H. Sayegh, X. G. Zheng, R. Peach, P. S. Linsley, L. A. Turka. 1996. Costimulatory function and expression of CD40 ligand, CD80, and CD86 in vascularized murine cardiac allograft rejection. Proc. Natl. Acad. Sci. USA 93: 13967-13972.
Hancock, W. W., F. M. Szaba, K. N. Berggren, M. A. Parent, I. K. Mullarky, J. Pearl, A. M. Cooper, K. H. Ely, D. L. Woodland, I. J. Kim, et al 2004. Intact type 1 immunity and immune-associated coagulative responses in mice lacking IFN--inducible fibrinogen-like protein 2. Proc. Natl. Acad. Sci. USA 101: 3005-3010.(Qin Ning2,*, Yi Sun3,*, M)
Immune coagulation is a major contributor to the pathogenesis of xenograft rejection, viral-induced hepatocellular injury and cytokine-induced fetal loss syndrome. In this study, we investigated the contribution of the novel gene product, fibrinogen-like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection. Using a mouse heterotopic cardiac transplant model, mouse fgl2(mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4- and CD8-positive T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Although mfgl2-deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2-deficient mice were prolonged and deposition of intravascular fibrin was diminished. Treatment of wild-type mice with a neutralizing anti-fgl2 Ab ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival. To address further the relevance of fgl2 in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. Human fgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8+ T cells, mature B cells (plasma cells), and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with human fgl2 expression. These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection.
Introduction
Fibrinogen-like protein 2 (fgl2)4/fibroleukin, also known as fgl2 prothrombinase, has recently been cloned and identified and shown to belong to the fibrinogen family of proteins. Mouse fgl2 (mfgl2) and human fgl2 (hfgl2) have been localized to chromosomes 5 and 7, respectively (1, 2, 3). fgl2 prothrombinase has been shown previously to have the attributes of a serine protease capable of directly cleaving prothrombin to thrombin leading to fibrin deposition (2). Several studies indicate that mfgl2 is involved in experimental xenograft rejection by mediating "immune coagulation," fibrin deposition, and microthrombus formation, leading to classical pathological changes of acute vascular rejection (4). The role of fgl2 in allorejection has not been examined in detail. Previously, Hancock et al. (5) have suggested that mfgl2 expression is correlated with allograft rejection, and strategies known to prevent allorejection including infusion of anti-CD154 prevent expression of mfgl2. However, this group recently also suggested that disruption of mfgl2 did not alter type 1 immunity or fibrin deposition associated with allograft rejection (6).
In this present study, we investigated the expression of fgl2 in acute allograft rejection, first using a mouse heterotopic cardiac transplant model, and subsequently extending our studies to examine hfgl2 expression in rejecting human renal allografts. Expression of fgl2 was studied by both in situ hybridization and immunochemistry. mfgl2 transcripts were highly expressed in macrophages, T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Treatment of mice with a high-titered neutralizing anti-fgl2 polyclonal Ab ameliorated the pathological injury and resulted in measurably increased graft survival. In rejecting human renal allografts, hfgl2 mRNA transcripts and protein expression correlated with the presence of rejection. Collectively, these studies suggest that fgl2 expression may be critical to the pathogenesis of both experimental and human allograft rejection and provide a rationale for targeting the fgl2 gene in an attempt to modulate allograft rejection.
Materials and Methods
Statistical analysis
Quantitative data were expressed as mean ± SD. Statistical analysis was conducted by using independent t test to compare effects of treatment on mean grade of rejection grade. Survival data were measured using a Kaplan-Meier model, and overall strata comparisons were made using log rank (Mantel-Cox) tests. The analysis was conducted on the statistical program for social sciences (SPSS), version 13.0, for Windows. A value of p < 0.05 was considered statistically significant.
Results
mfgl2 expression in rejecting grafts post-cervical heterotopic cardiac allotransplantation in mice
Hearts recovered from mice that underwent cervical heterotopic cardiac isogeneic or allogeneic transplantation at predetermined times posttransplantation (days 1, 3, 5, and 7) were examined for the presence of rejection (Table IV; Fig. 1, A–D) and expression of mfgl2. Transplantation of isogeneic grafts was used as controls. Expression of mfgl2 was examined at both the mRNA and protein levels by in situ hybridization and immunohistochemistry, respectively. Expression of mfgl2 was first detected at day 1 posttransplant, and expression of mfgl2 increased on days 3, 5, and 7 (Fig. 1, E–H and J–M). mfgl2 was seen primarily in infiltrating mononuclear cells and endothelial cells of the microvasculature, which correlated with the severity of the histopathological findings in rejecting grafts. There was little or no mfgl2 expression in grafts from the isogeneic group where there were no histological signs of rejection (Fig. 1, I–N).
Cellular source of mfgl2 and fibrin deposition in rejecting grafts in mice
By serial section staining, the majority of CD68+, CD4+, and CD8+ cells showed high expression of mfgl2 protein in rejecting grafts (Fig. 2, A–F). Fibrin deposition was also detected in the endothelium of microvascular vessels by immunoperoxidase staining by day 5 (Fig. 2, G and H).
Effects of fgl2 polyclonal Ab on the histopathological improvement in rejecting grafts post-cervical heterotopic cardiac allotransplantation in mice
The pathological grades of the rejecting cardiac grafts from mice treated with or without fgl2 polyclonal Ab based on a modification of the Banff criteria proposed by Billingham et al. (9) (Table II) are summarized in Table IV and Fig. 3. Grade of rejection of fgl2 Ab-treated grafts was statistically reduced when compared with untreated or control Ab-treated allografts on days 3, 5, and 7, whereas control Ab treatment had no effect on rejection grade (Fig. 3B). The histological changes within the rejecting cardiac grafts were improved post-fgl2 polyclonal treatment as shown in Fig. 3A, A–C, compared with that in Fig. 1, B–D, or Fig. 3AD, in which no Ab treatment or a control Ab was used.
Effect of fgl2 polyclonal Ab on survival of rejecting cardiac grafts post-cervical heterotopic heart allotransplantation in mice
Grafts from mice that had received two injections of 500 μg of fgl2 neutralizing polyclonal Ab had a modest, but statistically significant increase in survival compared with grafts from untreated mice or mice injected with a control polyclonal Ab by log rank (Mantel-Cox) analysis followed by a paired t test for individual groups. The mean survival time of Ab-treated mice was with a mean survival time of 9.4 ± 1.1 days compared with a survival time of 7.3 ± 0.5 days of untreated mice; and 7.3 ± 0.6 days of mice receiving control Ab (Fig. 4). Additionally, mice receiving two injections of Fab' equivalent to 500 μg of polyclonal anti-fgl2 (group E) were protected to the same degree as mice receiving the native anti-fgl2 molecule, with a mean survival of 9.7 + 1.3 days. These data are consistent with the hypothesis that protection occurs through neutralization of fgl2 rather than the involvement of other cells or molecules.
Heart grafts from BALB/cJ mice transplanted into mfgl2-deficient mice were universally rejected on day 6.4 ± 1.2, similar to transplantation of heart grafts from BALB/cJ mice, which were transplanted into fgl2+/+ littermate controls. In contrast, heart grafts from fgl2-deficient mice transplanted into BALB/cJ mice had a prolonged survival but were ultimately rejected on day 12.2 ± 1.4 (Table V).
Immunohistochemical assessment of hfgl2 expression in patients with renal acute allograft rejection
To address the relevance of hfgl2 in human acute allograft rejection, we studied patients with renal acute allograft rejection (Table III). Twenty-one of the patients were male, and 10 were female. The mean time to first rejection posttransplant was 14.9 + 24.1 mo. Grafted renal tissues with histopathological grades IB, IIA, IIB, and III were strongly and uniformly positive for hfgl2 expression, whereas IA lesions had less intense hfgl2 positivity, indicating a close association of hfgl2 expression with the histopathologic rejection and fibrin deposition. Expression of hfgl2 was seen primarily in renal tubule cells, in infiltrating mononuclear cells, and in the endothelium of small renal blood vessels and glomerular capillary wall in close proximity to fibrin deposits (Fig. 5).
By dual-staining immunohistochemistry, CD68+ (macrophages) and CD8+ (T cells) were shown to be the cellular source of hfgl2 (Fig. 6, A–D, respectively). Fibrin deposition was seen within the microvascular vessels, glomerular capillary wall and matrix, and the surface of infiltrating mononuclear cells in the blood vessels by immunoperoxidase staining in association with hfgl2 expression (Fig. 6, E and F).
Discussion
Fibrin deposition is a common element of many types of immunological reactions including delayed-type hypersensitivity, autoimmune disease, graft rejection, and the response to infectious agents (11, 12). Induction of procoagulant molecules on leukocytes and endothelial cells is thought to play an important role in the pathogenesis of thrombin generation and fibrin deposition at the sites of inflammation. This has been observed in the case of tissue factor, through the widespread constitutive expression of this molecule in subendothelial and perivascular tissues, suggesting that its major function is in initiating coagulation to achieve hemostasis after endothelial cell disruption.
Procoagulants other than tissue factor, which are induced specifically by immune mediators, may also play a critical role in regulating localized fibrin deposition. fgl2, also known as fibroleukin, is a 70-kDa type-2 transmembrane protein, which has been postulated to directly convert prothrombin to thrombin in the absence of factor VII or factor X (13). fgl2 has been shown previously to play a critical role in the pathogenesis of fulminant hepatitis induced by murine hepatitis virus strain 3 (MHV-3) (1, 7). Murine, porcine, and human fgl2 have now been cloned and sequenced, and fgl2 encodes the mouse fibrinogen-like protein (musfiblp), a previously described gene isolated from CTLs that shares significant homology to fibrinogen - and -chains (14, 15, 16). The murine and human proteins share 78% overall identity, with greater conservation at the C terminus. The C terminus of fgl2 corresponds to a highly conserved region that is found in fibrinogen as well as in other functionally unrelated fibrinogen-related proteins such as tenascin, ficolin, and angiopoietin (17, 18, 19).
In the context of innate immune activation, induction of fgl2 in macrophages and endothelial cells has been shown to contribute to the pathogenesis of tissue factor-independent fibrin deposition and organ injury. In support of its role as a coagulant are the observation that neutralizing Abs to mfgl2 prevent both fibrin deposition and death from MHV-3 infection (20). Recent studies have also shown that inhibition of reticuloendothelial cell mfgl2 expression through the use of gene-targeted fgl2-deficient (fgl2–/–) mice results in the prevention of MHV-3-induced fibrin deposition, liver injury, and death (7). Furthermore, we have recently reported that murine and human fgl2 prothrombinase/fibroleukin are highly expressed in endothelium, macrophages, and lymphocytes in xenograft rejection (4).
In the current study, elevated expression of mfg12 mRNA and protein was observed on vascular endothelial cells and infiltrating leukocytes, including macrophages, CD4- and CD8-positive T lymphocytes, in rejecting cardiac allografts in a mouse heterotopic cardiac transplant model, in association with deposits of fibrin. Treatment of mice with a high-titered neutralizing anti-mfgl2 polyclonal Ab reduced the pathological injury and led to modest but statistically significant increase in graft survival. The fact that Fab' anti-fgl2 was as protective as the native Ab suggests that the protection observed follows from neutralization of mfgl2 and is independent of other (FcR+) cells/molecules, including complement, in the environment. Furthermore, transplanted hearts from mfgl2–/– mice were largely devoid of fibrin deposition resulting in prolonged graft survival, whereas grafts from wild-type mice transplanted into mfgl2–/– recipients were rejected in a similar tempo, and with similar histopathology, as fgl2+/+ littermates. Collectively, these data provide evidence for a role for endothelial cell fgl2 expression in the fibrin deposition associated with allotransplant rejection and suggest that strategies designed to prevent endothelial cell fgl2 expression may prove of benefit to improving allotransplant graft survival. The data are further supported from patient studies in which increased fgl2 and fibrin expression were found mainly in renal tubule cells, infiltrating macrophages, CD8+ T cells, and plasmacytes, as well as endothelial cells in rejecting renal grafts.
Recent studies from Hancock et al. (6) have also examined the relevance of mfgl2 to fibrin deposition in allorejection. Their studies, similar to ours, showed that mfgl2–/– recipient mice rejected donor heart allografts in a fashion analogous to wild-type mice. Collectively, these studies support the hypothesis that (donor) endothelial cell production of mfgl2, rather than an immune-activated infiltrating leukocyte population, accounts for the fibrin deposition in allorejection (6). These data are further supported by our recent studies in xenotransplantation in which fibrin deposition associated with xenograft rejection was largely intravascular rather than associated with infiltrating inflammatory cells (21). Furthermore, xenografts from mfgl2–/– mice transplanted into rats were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium accounts for fibrin deposition of allo- and xenotransplant rejection.
It is important to appreciate that fgl2 along with other members of the fibrinogen family of molecules have also been implicated in delivery of signals, which activate various arms of the innate immune system (22). Accordingly, some of the effects mediated by alteration of fgl2 expression might reflect not simply altered thrombin deposition, but also an altered immune activation. As but one example, Chan et al. (23) reported that fgl2 modified expression of costimulatory molecules on developing dendritic cells, with a profound "downstream" effect on cytokine induction following allostimulation. Current studies from our laboratory support the hypothesis that an additional immunomodulatory role for fgl2 does indeed follow binding to a receptor expressed in multiple tissues (R. Liu, manuscript in preparation).
Although not the focus of this paper, the molecular pathways for induction of fgl2 have recently been studied. In murine hepatitis virus infection, nucleocapsid protein induces transcription of fgl2 through the transcription factor hepatic nuclear factor 4 and its cognate receptor (24, 25). In transplantation, fgl2 transcription appears to be regulated by cytokines. Macrophage induction of fgl2 is induced by IFN-, whereas preliminary data suggest that fgl2 transcription in endothelial cells occurs in response to TNF- but not IFN- (6). In contrast, induction of tissue factor is NF-B dependent, and, unlike fgl2, IFN- inhibits tissue factor transcription (M. F. Liu, unpublished data).
The importance of fgl2 to allograft rejection is supported first by the observation that expression of fgl2 correlates with rejection (6) and also by the fact that administration of neutralizing Ab to fgl2 diminished the pathological injury and improved graft survival in a similar fashion to that previously reported in murine hepatitis infection (20).
In the development of late interstitial fibrosis seen in cases of chronic rejection, a great deal of attention has been paid to the early fibrin deposition in the matrix in the rejected grafts. Acute rejection is known to be one of the high risk factors for this phenomenon (26, 27). An important observation in this current study in both the experimental mouse model and in humans is the finding of high expression of fgl2 prothrombinase in infiltrating cells within the matrix or interstitial region of the kidney. Interestingly, the expression of fgl2 was in seen in close association with local fibrin deposition, consistent with a role for fgl2 in the pathogenesis of chronic allograft rejection, although further studies are necessary to define a role for fgl2 in chronic rejection.
In summary, these studies in both a murine model and in human renal allograft rejection provide evidence suggestive of a role for fgl2 in the thrombosis associated with acute allograft rejection. The data indicate that endothelial cells rather than leukocyte fgl2 expression accounts for intravascular fibrin deposition. Although not part of this study, data generated by our group and that of Hancock et al. suggest that one mechanism of altered fgl2 transcription is through elaboration of cytokines including IFN- and TNF, although additional studies are necessary to determine whether other cytokines are involved in the induction of fgl2. The elaboration of fgl2 during acute allograft rejection and the interstitial fibrin deposition may lead to further injury and development of chronic allograft rejection, but additional studies are required to firmly establish the role of fgl2 in this process, and in particular a potential immunomodulatory role for fgl2 in this process. These studies provide a rationale to target fgl2 for therapeutic intervention in an attempt to ameliorate both acute and chronic allograft rejection.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the National Science Fund for Distinguished Young Investigators (30225040 (to Q.N.), 30123019 (to X.L.)) from the Natural Science Foundation of China (NSFC), NSFC Operating Fund 30100171 and 30170846, National Key Basic Research Program of China (2001CB510008), and the Canadian Institutes for Health Research Grant FRN33780
2 Address correspondence and reprint requests to Dr. Qin Ning, Laboratory of Infectious Immunology and Department of Infectious Disease, Tongji Hospital, 1095 Jie Fang Avenue, Wuhan 430030, China. E-mail address: qning{at}tjh.tjmu.edu.cn
3 Current address: Department of Immunology, Medical College of Chinese People’s Armed Police Force, Tianjin 300162, China.
4 Abbreviations used in this paper: fgl2, fibrinogen-like protein 2; mfgl2, mouse fgl2; hfgl2, human fgl2; Dig, digoxigenin; AP, alkaline phosphatase; MHV-3, murine hepatitis virus strain 3.
Received for publication August 31, 2004. Accepted for publication March 4, 2005.
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