MMP (Matrix Metalloprotease)-9-Producing Monocytes Enable T Cells to Invade the Vessel Wall and Cause Vasculitis - PubMed (original) (raw)

MMP (Matrix Metalloprotease)-9-Producing Monocytes Enable T Cells to Invade the Vessel Wall and Cause Vasculitis

Ryu Watanabe et al. Circ Res. 2018.

Abstract

Rationale: Giant cell arteritis (GCA)-a primary vasculitis of medium and large arteries-is associated with vessel wall damage, elastic membrane fragmentation, and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury.

Objective: The MMP (matrix metalloproteinase)-9-a type IV collagenase-is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent.

Methods and results: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts, and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from patients with GCA. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD (cluster of differentiation) 4+ T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID (severe combined immunodeficiency) chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting rMMP-9 (recombinant MMP-9). Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates ( P<0.001), reduced intramural neoangiogenesis ( P<0.001), and prevented intimal layer hyperplasia ( P<0.001). rMMP-9 amplified all domains of vasculitic activity, promoted assembly of T-cell infiltrates ( P<0.05), intensified formation of new microvessels ( P<0.001), and worsened intimal thickening ( P<0.001). Systemic delivery of N-acetyl-proline-glycine-proline-a matrikine produced by MMP-9-mediated gelatinolysis-had limited vasculitogenic effects.

Conclusions: In large vessel vasculitis, MMP-9 controls the access of monocytes and T cells to the vascular wall. T cells depend on MMP-9-producing monocytes to pass through collagen IV-containing basement membrane. Invasion of vasculitogenic T cells and monocytes, formation of neoangiogenic networks, and neointimal growth all require the enzymatic activity of MMP-9, identifying this protease as a potential therapeutic target to restore the immunoprivilege of the arterial wall in large vessel vasculitis.

Keywords: T lymphocytes; basement membrane; giant cell arteritis; macrophages; matrix metalloproteinases.

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Figures

Figure 1.. MMP-9-producing monocytes and macrophages in GCA.

(A) Biopsies from GCA-affected temporal arteries and from non-inflamed arteries were processed for quantification of MMP-9 transcripts by RT-PCR. Mean ± SEM from 10 tissue samples. (B) Immunostaining of tissues section from GCA temporal arteries. Pro-MMP9; brown. Scale bar, 100 μm. (C) Dual-color immunostaining of GCA-affected temporal artery sections. Pro-MMP-9; green. Macrophage marker CD68; red. Pro-MMP-9+ CD68+ cells are yellow. Scale bar indicates 100 μm. (D-F) Dual-color immunostaining of GCA-affected temporal artery sections. Pro-MMP-9; red. Macrophage marker PU.1; nuclei dark grey. (E, F) Pro-MMP-9+ PU.1+ macrophages and multinucleated giant cells along the fragmented lamina elastica. Magnified view, X400. (G, H) Dual-color immunostaining of pro-MMP9 (red) and PU.1 (nuclei dark grey) in tissues section from GCA aortitis showing granulomatous infiltrate with multinucleated giant cells surrounding necrotic medial layer. G; X100. H, X400. Scale bar indicates 100 μm. (I) Monocytes were isolated from freshly harvested peripheral blood of 6 GCA patients. Monocyte-derived macrophages (n=6) were differentiated with M-CSF for 5 days and stimulated with LPS/IFN-γ for 24 hrs. MMP transcripts were measured by RT-PCR. Data are mean ± SEM. Rel. Exp.; Relative expression. After adjustment for multiple testing using the Benjamin-Hochberg method, the comparisons of MMP-2 and MMP-9 are statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. Mann-Whitney test (A) and two-way ANOVA (F).

Figure 1.. MMP-9-producing monocytes and macrophages in GCA.

Figure 2.. MMP-9+ monocytes enable T cell invasion through basement membrane.

(A) Artificial basement membranes were built by coating collagen IV and collagen I layers on top of membranes (pores 8.0 μm). Purified T cells, monocytes or T cell/monocyte mixtures were layered on top of the collagen. Invasive capacity of T cells and monocytes was quantified by enumerating the cells in the lower chamber. (B, C) Monocytes (100,000/insert, B) and CD4+ T cells (100,000/insert, C) were isolated from healthy controls and GCA patients (n=8, each) and stimulated with LPS/IFN-γ and anti-CD3/CD28, respectively. After 24 hrs, cells that had passed through collagen IV/I were counted. Unpaired 2-tailed Student’s t test. (D) CD4+ T cell/monocyte mixtures (100,000 each) were placed on top of the collagen gel. In each well, isotype control IgG or anti-MMP-9 antibody (10 μg/ml) was added. After 24 hours, T cells which had passed though the gels were counted (n=8, each). Two-way ANOVA. All data are mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3.. Blocking MMP-9 protects the arterial wall from inflammatory damage.

Human arteries were engrafted into NSG mice and vasculitis was induced by adoptive transfer of peripheral blood mononuclear cells of GCA patients. Chimeric mice were treated with either anti-MMP-9 or control IgG and vessel wall inflammation was assessed in explanted artery grafts. (A) H&E-stained tissue sections. Scale bars, 200 μm. (B) Density of the T-cell infiltrate assessed by quantifying tissue T-cell receptor (TCR) transcripts by RT-PCR (n = 10). (C) Immunohistochemical staining of CD3+ T cells in tissue sections. Scale bar, 100 μm. (D) Enumeration of CD3+ T cells in tissue sections from 10 artery grafts treated with control IgG or anti-MMP-9. Data from paired grafts are connected by a line. (E) Tissue transcriptome analysis for the macrophage marker CD163, the innate cytokines (IL-1β, IL-6 and TNF-α), and T-cell effector cytokines IFN-γ, IL-17 and IL-21 in control-IgG and anti-MMP-9-treated grafts. Results from 10 arteries in each treatment arm are displayed as heat maps with Log2 scale. After adjustment for multiple comparisons, differences in CD163, IL-1β, IL-6, IFN-γ and IL-21 expression were statistically significant. (F) Explanted artery grafts were digested and CD4+ T cells were identified by flow cytometric analysis. Absolute numbers /200 mg tissue are presented. (G, H) Intracellular cytokines (IFN-γ and IL-21) were analyzed in tissue-extracted T cells by flow cytometry. Frequencies (G) and absolute numbers (H) of IFN-γ-producing and IL-21-producing T cells from 8 grafts in each arm are shown. All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Rel. Exp.; Relative expression. Paired 2-tailed Student’s t test.

Figure 4.. Anti-MMP-9 Ab inhibits inflammation-induced neoangiogenesis and intimal hyperplasia.

Vasculitis was induced in engrafted human arteries and chimeric mice were treated with anti-MMP-9 antibodies as in Figure 3. (A) H&E-stained tissue sections from control IgG and anti-MMP-9 treated arteries showing intimal thickening. Representative images. Black arrowheads mark the lamina elastic interna (LEI), and red arrows span the distance from the LEI to the lumen. Scale bar, 100 μm. (B) Measurement of the intimal layer thickness in 10 grafts treated with control IgG or anti-MMP-9. Lines connect data from paired arteries. (C) Immunostaining of microvessels in the arterial wall. Vascular lumina visualized as CD31+α-SMA+ structures. Scale bar, 100 μm. (D) Density of microvessels in paired arterial grafts treated with control IgG or anti-MMP-9 measured by dual-color immunohistochemistry as in C. Data from 10 paired arterial grafts. (E) Gene expression profiling for growth factors measured by RT-PCR. Mean ± SEM from 8 samples in each treatment arm. *p < 0.05, ***p < 0.001. Paired 2-tailed Student’s t test.

Figure 5.. Excess MMP-9 drives vessel wall inflammation.

NSG mice were engrafted with human arteries and reconstituted with PBMC from GCA patients to induce vasculitis as in Figure 3. Mice were injected with vehicle, enzymatically inactive MMP-9 (iMMP-9, 600 μg over 2 weeks) or recombinant MMP-9 (rMMP-9; 600 μg over 2 weeks) and human arteries were explanted for histology and transcriptome analysis. (A) H&E-stained tissue sections of explanted arteries demonstrating rMMP-9-induced exacerbation of vasculitis. Scale bar, 200 μm. (B) Density of wall-residing T cells assessed by RT-PCR of TCR transcripts. Mean ± SEM from 8 artery grafts. (C) Wall-infiltrating T cells visualized by immunolabeling with anti-CD3. Scale bar, 100 μm. (D) Density of the T cell infiltrates determined by enumerating wall-infiltrating CD3+ T cells in tissue sections from 8 arteries treated with iMMP-9 or rMMP-9. Results from paired grafts connected by a line. (E, F) Gene expression of CD163, IL-1β, IL-6, TNF-α, IFN-γ, IL-17 and IL-21 in artery explants after treatment with vehicle, iMMP-9 or rMMP-9. Data from 8 samples in each treatment arm. After adjustment for multiple comparisons, CD163, IL-1β, IL-6, IFN-γ and IL-21 remain statistically significant. All data are mean ± SEM. *p < 0.05, **p < 0.01. Rel. Exp.; Relative expression. Paired 2-tailed Student’s t test.

Figure 6.. MMP-9 overexpression amplifies inflammation-induced neoangiogenesis and outgrowth of the intimal layer.

Vasculitis was induced in human artery-NSG mice and chimeras were assigned to the vehicle, iMMP-9 or rMMP-9 treatment group as in Figure 5. Intimal thickness was measured as the distance between the lamina elastic interna and the luminal endothelial lining. (A) H&E-stained tissue sections from explanted arteries. Representative images. Black arrowheads mark the LEI and the red arrow spans the thickness of the intimal layer. Scale bar, 100 μm. (B) Intimal layer thickness in 8 arterial grafts treated with either vehicle, iMMP-9 or rMMP-9. Lines connect data from paired samples. (C) CD31+α-SMA+ microvessels identified by dual-color immunostaining. Nuclei marked by DAPI. Representative images from iMMP-9 and rMMP-9 treated tissues. Scale bars, 100 μm. (D) Densities of microvessels in paired arterial grafts treated with rMMP-9 or iMMP-9 measured as in C. Data from 8 paired arterial grafts are connected by a line. (E) Tissue transcriptome of angiogenic cytokines in the explants from iMMP-9 and rMMP-9-treated mice. Results from 6 samples in each treatment arm. All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Rel. Exp.; Relative expression. Paired 2-tailed Student’s t test.

Figure 7.. Limited vasculitogenic effects of the matrikine Ac-PGP.

Vasculitis was induced by transferring PBMC from GCA patients into NSG mice previously engrafted with human arteries as in Fig.3. Chimeras were subsequently treated with either vehicle or N-ac-PGP (ac-PGP, 600 μg over 2 weeks). Explanted arteries were examined for histology and transcriptome analysis. (A) H&E-stained tissue sections from artery grafts in both treatment arms. Scale bar, 200 μm. (B) TCR transcripts measured by RT-PCR in the explanted artery tissues (n = 8, each). (C) Immunostaining of wall-infiltrating CD3+ T cells. Scale bar, 100 μm. (D) Quantification of tissue-resident CD3+ T cells. Lines connect data in 8 paired samples. (E) H&E-stained tissue sections from the explanted artery tissues. Scale bar, 100 μm. (F) Measurement of intimal layer thickness in 8 paired arterial grafts. Black arrowheads mark the lamina elastic interna and red arrow spans the thickness of the intimal layer. (G) Immunohistochemistry of CD31+α-SMA+ microvessels. Representative images from both treatment arms. Scale bar, 100 μm. (H) Microvascular density in paired arterial grafts measured by dual-color immunohistochemistry as in G. Data from 8 paired arterial grafts are connected by a line. All data are mean ± SEM. *p < 0.05. Rel. Exp.; Relative expression. Paired 2-tailed Student’s t test.

Comment in

Monocytes M( MP)aking Way for T-Cell Vascular Infiltration.
Nosalski R, Maffia P, Guzik TJ. Nosalski R, et al. Circ Res. 2018 Aug 31;123(6):638-640. doi: 10.1161/CIRCRESAHA.118.313668. Circ Res. 2018. PMID: 30355241 No abstract available.

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MMP (Matrix Metalloprotease)-9-Producing Monocytes Enable T Cells to Invade the Vessel Wall and Cause Vasculitis - PubMed (original) (raw)