Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep;114(6):846-56.
doi: 10.1172/JCI21767.

T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase

Kian Peng Koh  1 , Yinong Wang, Tai Yi, Stephen L Shiao, Marc I Lorber, William C Sessa, George Tellides, Jordan S Pober
Affiliations

T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase

Kian Peng Koh et al. J Clin Invest. 2004 Sep.

Abstract

Allograft vascular dysfunction predisposes to arteriosclerosis and graft loss. We examined how dysfunction develops in transplanted human arteries in response to circulating allogeneic T cells in vivo using immunodeficient murine hosts. Within 7-9 days, transplanted arteries developed endothelial cell (EC) dysfunction but remained sensitive to exogenous NO. By 2 weeks, the grafts developed impaired contractility and desensitization to NO, both signs of VSMC dysfunction. These T cell-dependent changes correlated with loss of eNOS and expression of iNOS--the latter predominantly within infiltrating T cells. Neutralizing IFN-gamma completely prevented both vascular dysfunction and changes in NOS expression; neutralizing TNF reduced IFN-gamma production and partially prevented dysfunction. Inhibiting iNOS partially preserved responses to NO at 2 weeks and reduced graft intimal expansion after 4 weeks in vivo. In vitro, memory CD4+ T cells acted on allogeneic cultured ECs to reduce eNOS activity and expression of protein and mRNA. These effects required T cell activation by class II MHC antigens and costimulators (principally lymphocyte function-associated antigen-3, or LFA-3) on the ECs and were mediated by production of soluble mediators including IFN-gamma and TNF. We conclude that IFN-gamma is a central mediator of vascular dysfunction and, through dysregulation of NOS expression, links early dysfunction with late arteriosclerosis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effects of allogeneic T cells on arterial graft function at 1 week in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) 7–9 days before harvest (n = 5 pairs from four experiments). (A–D) Response curves. Restriction response to various concentrations of PGF2α (A) and relaxation response curves for nitroprusside (B), bradykinin (C), or substance P (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. mN, milliNewtons. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD45, and HLA-DR. The staining for human CD45 is indistinguishable from that for human CD3 (data not shown). Original magnification, ×200.
Figure 2
Figure 2
Effects of allogeneic T cells on arterial graft function at 2 weeks in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) for 13–15 days (n = 4–7 pairs from four to six experiments). (A–D) Response curves. Constriction response to various concentrations of PGF2α (A) and relaxation response to bradykinin (B), nitroprusside (C), or YC-1 (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD3, and iNOS. iNOS expression was highly localized to infiltrating T cells (arrows). Original magnification: CD31 and CD3 panels, ×200; iNOS panels, ×400.
Figure 3
Figure 3
Effects of IFN-γ neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti–IFN-γ mAb (filled triangles) or control mAb (IgG2a) (open triangles) for 2 weeks (n = 5 matched triplicates from three experiments). (A and B) Endothelium-independent responses to various concentrations of PGF2α (A) and nitroprusside (B). (C and D) Endothelium-dependent response to bradykinin before (C) and after (D) treatment with L-NAME. *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG2a group, n = 3–5. (E) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. The luminal HLA-DR–positive cells are also human CD31–positive (data not shown). Goat anti-mouse secondary Ab alone did not stain the grafts. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (F and G) RNA transcript levels of iNOS (F) and eNOS (G) in whole tissue sections analyzed by quantitative RT-PCR.
Figure 4
Figure 4
Effects of TNF neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti-TNF mAb (filled triangles) or control mAb (IgG1) (open triangles) for 2 weeks (n = 4 matched triplicates from two experiments). (A–C) Concentration-dependent responses to PGF2α (A), nitroprusside (B), and bradykinin (C). *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG1 group; n = 3–4. (D) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (E and F) RNA transcript levels of iNOS (E) and eNOS (F) in whole tissue sections analyzed by quantitative RT-PCR.
Figure 5
Figure 5
Contribution of iNOS to VSMC dysfunction and graft arteriosclerosis. (A–C) Arterial grafts recovered from animals at 2 weeks were exposed to the iNOS inhibitor 1400W ex vivo in the bath chamber (n = 3–6 pairs from four to five experiments). Concentration-dependent responses to PGF2α (A), bradykinin (B), and nitroprusside (C) of grafts from saline (squares) and PBMC (triangles) groups were measured before (open symbols) and after (filled symbols) treatment with 1400W. *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC group before 1400W treatment. Treatment with 1400W did not cause significant changes in any response of the arteries harvested from saline-treated animals. Saline group after 1400W treatment is not shown in the nitroprusside response. (D) Arterial grafts were recovered from mice reconstituted with PBMCs and injected daily with saline (open squares) or 1400W (filled squares) for 2 weeks (n = 5 pairs from three experiments). Smooth muscle function was assessed by the relaxation response of preconstricted arteries to various concentrations of nitroprusside. (E and F) Paired arterial grafts recovered from mice reconstituted with PBMCs and injected daily with saline or 1400W for 4 weeks (n = 6 pairs from three experiments). Morphometry of H&E-stained tissue sections (E), in which compartments are demarcated as adventitia (AD), media (M), and intima (I), was analyzed to calculate mean intimal thickness (F). Original magnification: ×200.
Figure 6
Figure 6
CD4+ memory T cells downregulate eNOS in class II MHC–positive ECs in vitro. (A) Flow cytometry of resting (shaded) or IFN-γ–pretreated (bold line) HUVECs immunostained for surface class I MHC (HLA-ABC), class II MHC (HLA-DR), and ICAM-1 (CD54). Dotted line, IgG1 control. (B) NOS activity of HUVECs, either untreated or pretreated with IFN-γ, after CD4+ T cell coculture. (C) NOS activity of IFN-γ–pretreated HUVECs after CD8+ or CD4+ T cell coculture. (D) NOS activity of IFN-γ–pretreated HUVECs after CD4+ T cell coculture with control or anti–HLA-DR mAb. Below: eNOS protein levels determined by Western blot and densitometry and normalized to levels of β-actin in arbitrary units. Data in A–D represent mean ± SEM from at least four experiments. (E) eNOS RNA levels in IFN-γ–pretreated HUVECs after CD4+ T cell coculture with control IgG, or inhibitory mAb against HLA-DR, LFA-3, CD2, and CD40L (CD154). Values were expressed relative to those of HUVECs alone. Isotype-matched IgG controls were pooled. Data represent mean ± SEM from five to eight experiments. #P < 0.001 vs. ECs alone; *P < 0.01 and **P < 0.001 vs. CD4+ T cell + IgG group. (F) Epifluorescence of eNOS cellular localization (green) with nuclei costaining (blue) in untreated or IFN-γ–pretreated HUVECs, cocultured with CD4+ T cells. IFN-γ–pretreated HUVECs were also cocultured with subsets of CD4+/CD45RA+ (naive) cells and CD4+/CD45RO+ (memory) cells. The smaller nuclei belong to T cells. Images are representative of three experiments. All cocultures were incubated for 3 days.
Figure 7
Figure 7
Contact dependence of EC response to T cells. Epifluorescence of eNOS immunostaining (green) with nuclei costaining (blue) of HUVECs that were pretreated with IFN-γ for 3 days prior to coculture with CD4+ T cells separated by a transwell. (A) ECs and T cells were cocultured in the absence of contact. (B) ECs below a transwell containing T cells in contact with IFN-γ–pretreated ECs. (C) ECs below a transwell containing T cells in contact with untreated (resting) ECs.
Figure 8
Figure 8
IFN-γ and TNF act in concert to reduce eNOS expression. (A) Flow cytometry of HUVECs transduced with β-gal (shaded) or class II transactivator (bold line) and stained for surface class I MHC (HLA-ABC), class II MHC (HLA-DR), and ICAM-1 (CD54). (B) Epifluorescence of eNOS cellular localization (green) with nuclei costaining (blue) in β-gal– or CIITA-transduced HUVECs cocultured for 3 days with CD4+ T cells. CIITA-transduced HUVECs were also cocultured with CD4+ T cells in the presence of anti-TNF and anti–IFN-γ mAb alone or in combination. mAb control: HUVEC cultures treated with both anti-TNF and anti–IFN-γ mAb and stained with secondary Ab only. Images are representative of five experiments.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Libby P, Pober JS. Chronic rejection. Immunity. 2001;14:387–397. - PubMed
    1. Pethig K, Heublein B, Wahlers T, Haverich A. Mechanism of luminal narrowing in cardiac allograft vasculopathy: inadequate vascular remodeling rather than intimal hyperplasia is the major predictor of coronary artery stenosis. Working Group on Cardiac Allograft Vasculopathy. Am. Heart J. 1998;135:628–633. - PubMed
    1. Fish RD, et al. Responses of coronary arteries of cardiac transplant patients to acetylcholine. J. Clin. Invest. 1988;81:21–31. - PMC - PubMed
    1. Tullius SG, Tilney NL. Both alloantigen-dependent and -independent factors influence chronic allograft rejection. Transplantation. 1995;59:313–318. - PubMed
    1. Davis SF, et al. Early endothelial dysfunction predicts the development of transplant coronary artery disease at 1 year posttransplant. Circulation. 1996;93:457–462. - PubMed

Publication types

MeSH terms