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Graft arteriosclerosis: molecular features of macrophage activation
Graft Arteriosclerosis:
Molecular
Features of Macrophage
Activation
M.E.
G
RAFT arteriosclerosis is a key feature of most chronic rejection syndromes.’ The classic feature is a conccntric arterial thickening that sclectivcly involves donor vessels. In humans, pathologic studies of transplant vessels have shown that graft arteriosclerosis occurs with high prcvalcncc. develops at an accelerated pace. and involves a spectrum of vessels in a longitudinal fashion. suggesting it is mediated by an alloimmunc injury.’ Experimental rodent transplant models have provided insight regarding the pivotal role of the alloantigen response and interaction of alloantigen-independent factors on development of graft arteriosclerosis. The features common among the various transplant models include pcrivascular infiltrates, luminal adhesion of mononuclear cells, and subluminal accumulation of inflammatory and myointimal cells. Immunostaining typically demonstrates that the cxpandcd intima is rich in macrophagcs. T cells, and smooth muscle cells. These fcaturcs appear preserved in spite of species and organ ditfcrcnccs across models of graft arteriosclerosis. The working hypothesis in my laboratory is that the macrophagc activated within the cytokine-rich environment of the transplanted heart mediates the arteriosclerotic thickening. The acquisition of functions by activated macrophages is typically conferred by production of gent products which in turn arc due to alterations in gene regulation. One of our goals has been to determine the activation products produced by macrophagcs in transplant artcriosclcrosis. Macrophages can be altered by isolation and culturing; therefore WC study the activation phenotype in vivo-directly in the pathophysiologic environment in which transplant arteriosclerosis develops. Thus, we have prcscrvcd both the r&dent and infiltrating cell populations, and the inductive and suppressive stimuli likely to regulate the macrophagc. Our investigations have examined known Factors asaociatcd with macrophage activation (chcmokincs, cytokinc and growth factors) and sought to identif’y novel macrophagc molecules. IDENTIFICATION OF NOVEL MACROPHAGE
cDNA
WC adapted differential mRNA display to identify genes whose cxprcssion was incrcascd in cardiac allografts in the macrophage-rich stage of transplant arteriosclerosis in comparison with cardiac syngrafts. Thcsc studies culminated in the isolation of 3 indcpendcnt cDNA fragments exprcsscd by macrophages.3 Two of these wcrc novel and have been
termed allograft inflammatory factors- 1 and -2 (AIF- 1 and AIF-2); the third is Gal/GalNAc macrophage Icctin. a carbohydrate receptor. Temporal studies demonstrated that gene transcript levels increase by day 7 and remain elevated through day 120, as might be expected with an ongoing inflammatory stimulation. Full length cDNA clones for AIF- and Gal/GalNAc macrophage lectin have been obtained by screening a rat cardiac allograft cDNA library,‘,’ whereas PCR-based cloning systems are being used to isolate 5’ portions of the AIF- cDNA. By Northern blot analysis, WC learned that these factors are selectively expressed by mycloid cells (monocytcs and neutrophils) compared with other cells and organs studied. For all 3 of these factors. expression localized to mononuclear cells infiltrating the heart as evidenced by in situ hybridization or immunohistochcmical techniques.
IN VITRO
REGULATION
OF MACROPHAGE
FACTORS
Using cultured macrophage populations we have demonstrated that all 3 factors arc regulated by various factors associated with T cell activation. Induction of GaliGalNAc macrophage lectin induction was evident only when Concanavalin A activated T cells or their supernatants were co-cultured with various macrophagc populations.” Exogenous intcrleukin-l/3. -2, -4, -6 or interferon-y alone or in combination failed to cause upregulation of this Icctin, indicating that it is tightly regulated. Interferon-y produced a late uprcgulation of AIF-I but an early induction of AIF-2.’
EXPRESSION OF MACROPHAGE HEART TRANSPLANTS
FACTORS
IN HUMAN
Preliminary in situ hybridization studies have demonstrated that Gal/GalNAc macrophagc lectin, AIF- 1. and AIFtranscripts arc expressed by subsets of mononuclear cells within the neointimal and perivascular space of human transplant vessels. By RT-PCR, immunoblotting, and im-
From the Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts. Address reprint requests to Dr M.E. Russell, Harvard School of Public Health, Cardiovascular Biology Laboratory, 677 Huntington Avenue, Boston, MA 02115.
0041-1345/97/$17.00 PII soo41-1345(97)00499-5
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2542
Transplantation
Proceedings,
29, 2542-2543
(1997)
2543
GRAFT ARTERIOSCLEROSIS
munocytochemistry, AIF- antgen is present at high levels in human transplanted hearts compared with hearts with dilated cardiomyopathy, atria1 tissue, and unrelated donor aortic tissue.’ As in the rat, AIF- also localizes to a subset of macrophages. In a collaborative study with Dr Bruce McManus, we have now demonstrated that AIF- is present in a series of human transplant vessels. Hence, we have demonstrated that one can use rodent models of graft arteriosclerosis to gain insight into macrophage activation, correlate with arteriosclerotic thickening, and identification and isolation of novel macrophage factors. Our current goal is to establish the in vivo and in vitro conditions that regulate these macrophage factors and to determine their functional properties in macrophage activation. Ultimately, we plan to determine if manipulations
designed to modulate levels of these three macrophagederived molecules will reduce arteriosclerotic development.
REFERENCES 1. Paul LC, Fellstrijm
B: Transplantation
2. Tullius SG, Tilney NL: Transplantation
53:1169,
1992
59:313,
1995
3. Utans U, Liang P, Wyner LR. et al: Proc Nat1 Acad Sci USA 91~6463, 1994 4. Russell ME, Utans U, Wallace 1994
AF, et al: J Clin Invest 04:722,
5. Utans U, Arceci RJ, Yamashita 1995
Y, et al: J Clin Invest 95:2954,
6. Utans 1996
U, Quist W, Wilson
J, et al: Transplantation
61:1387,
Molecular
Features of Macrophage
Activation
M.E.
G
RAFT arteriosclerosis is a key feature of most chronic rejection syndromes.’ The classic feature is a conccntric arterial thickening that sclectivcly involves donor vessels. In humans, pathologic studies of transplant vessels have shown that graft arteriosclerosis occurs with high prcvalcncc. develops at an accelerated pace. and involves a spectrum of vessels in a longitudinal fashion. suggesting it is mediated by an alloimmunc injury.’ Experimental rodent transplant models have provided insight regarding the pivotal role of the alloantigen response and interaction of alloantigen-independent factors on development of graft arteriosclerosis. The features common among the various transplant models include pcrivascular infiltrates, luminal adhesion of mononuclear cells, and subluminal accumulation of inflammatory and myointimal cells. Immunostaining typically demonstrates that the cxpandcd intima is rich in macrophagcs. T cells, and smooth muscle cells. These fcaturcs appear preserved in spite of species and organ ditfcrcnccs across models of graft arteriosclerosis. The working hypothesis in my laboratory is that the macrophagc activated within the cytokine-rich environment of the transplanted heart mediates the arteriosclerotic thickening. The acquisition of functions by activated macrophages is typically conferred by production of gent products which in turn arc due to alterations in gene regulation. One of our goals has been to determine the activation products produced by macrophagcs in transplant artcriosclcrosis. Macrophages can be altered by isolation and culturing; therefore WC study the activation phenotype in vivo-directly in the pathophysiologic environment in which transplant arteriosclerosis develops. Thus, we have prcscrvcd both the r&dent and infiltrating cell populations, and the inductive and suppressive stimuli likely to regulate the macrophagc. Our investigations have examined known Factors asaociatcd with macrophage activation (chcmokincs, cytokinc and growth factors) and sought to identif’y novel macrophagc molecules. IDENTIFICATION OF NOVEL MACROPHAGE
cDNA
WC adapted differential mRNA display to identify genes whose cxprcssion was incrcascd in cardiac allografts in the macrophage-rich stage of transplant arteriosclerosis in comparison with cardiac syngrafts. Thcsc studies culminated in the isolation of 3 indcpendcnt cDNA fragments exprcsscd by macrophages.3 Two of these wcrc novel and have been
termed allograft inflammatory factors- 1 and -2 (AIF- 1 and AIF-2); the third is Gal/GalNAc macrophage Icctin. a carbohydrate receptor. Temporal studies demonstrated that gene transcript levels increase by day 7 and remain elevated through day 120, as might be expected with an ongoing inflammatory stimulation. Full length cDNA clones for AIF- and Gal/GalNAc macrophage lectin have been obtained by screening a rat cardiac allograft cDNA library,‘,’ whereas PCR-based cloning systems are being used to isolate 5’ portions of the AIF- cDNA. By Northern blot analysis, WC learned that these factors are selectively expressed by mycloid cells (monocytcs and neutrophils) compared with other cells and organs studied. For all 3 of these factors. expression localized to mononuclear cells infiltrating the heart as evidenced by in situ hybridization or immunohistochcmical techniques.
IN VITRO
REGULATION
OF MACROPHAGE
FACTORS
Using cultured macrophage populations we have demonstrated that all 3 factors arc regulated by various factors associated with T cell activation. Induction of GaliGalNAc macrophage lectin induction was evident only when Concanavalin A activated T cells or their supernatants were co-cultured with various macrophagc populations.” Exogenous intcrleukin-l/3. -2, -4, -6 or interferon-y alone or in combination failed to cause upregulation of this Icctin, indicating that it is tightly regulated. Interferon-y produced a late uprcgulation of AIF-I but an early induction of AIF-2.’
EXPRESSION OF MACROPHAGE HEART TRANSPLANTS
FACTORS
IN HUMAN
Preliminary in situ hybridization studies have demonstrated that Gal/GalNAc macrophagc lectin, AIF- 1. and AIFtranscripts arc expressed by subsets of mononuclear cells within the neointimal and perivascular space of human transplant vessels. By RT-PCR, immunoblotting, and im-
From the Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts. Address reprint requests to Dr M.E. Russell, Harvard School of Public Health, Cardiovascular Biology Laboratory, 677 Huntington Avenue, Boston, MA 02115.
0041-1345/97/$17.00 PII soo41-1345(97)00499-5
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2542
Transplantation
Proceedings,
29, 2542-2543
(1997)
2543
GRAFT ARTERIOSCLEROSIS
munocytochemistry, AIF- antgen is present at high levels in human transplanted hearts compared with hearts with dilated cardiomyopathy, atria1 tissue, and unrelated donor aortic tissue.’ As in the rat, AIF- also localizes to a subset of macrophages. In a collaborative study with Dr Bruce McManus, we have now demonstrated that AIF- is present in a series of human transplant vessels. Hence, we have demonstrated that one can use rodent models of graft arteriosclerosis to gain insight into macrophage activation, correlate with arteriosclerotic thickening, and identification and isolation of novel macrophage factors. Our current goal is to establish the in vivo and in vitro conditions that regulate these macrophage factors and to determine their functional properties in macrophage activation. Ultimately, we plan to determine if manipulations
designed to modulate levels of these three macrophagederived molecules will reduce arteriosclerotic development.
REFERENCES 1. Paul LC, Fellstrijm
B: Transplantation
2. Tullius SG, Tilney NL: Transplantation
53:1169,
1992
59:313,
1995
3. Utans U, Liang P, Wyner LR. et al: Proc Nat1 Acad Sci USA 91~6463, 1994 4. Russell ME, Utans U, Wallace 1994
AF, et al: J Clin Invest 04:722,
5. Utans U, Arceci RJ, Yamashita 1995
Y, et al: J Clin Invest 95:2954,
6. Utans 1996
U, Quist W, Wilson
J, et al: Transplantation
61:1387,