Role of the T cell in the genesis of angiotensin II-induced hypertension and vascular dysfunction methods
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Effect of cytokine blockade on angiotensin II-induced hypertension
Role of cytokines in blood pressure regulation and vascular O 2 ·- production. (A) Production of TNF-α (top) and IFN-γ (bottom) measured by cytometric bead array from anti-CD3-stimulated peripheral blood T cells isolated from C57BL/6 and p47 phox-/- mice infused with saline (sham) or angiotensin II for 14 d ( n = 8 in each group). (B) Anti-CD3-stimulated production of TNF-α from spleen-derived T cells in response to in vitro coincubation without (vehicle; n = 6) or with 100 nm angiotensin II (Ang II; n = 6). (C) Noninvasive blood pressure measurements at baseline and during angiotensin II infusion measured by tail cuff in mice injected IP with control IgG ( n = 4) or with anti-TNFα therapy (etanercept, ETA; 8 mg/kg; n = 6) 3 d before and every 3 d throughout the experiment. (D) Aortic O 2 ·- levels after angiotensin II...
MATERIALS AND METHODS
C57BL/6, RAG-1 -/-, and AT1a -/- mice were obtained from Jackson ImmunoResearch Laboratories. The p47 phox -/- mice and their appropriate controls were obtained from Taconic. All experimental protocols were approved by the institutional Animal Care and Use Committee at Emory University. All mice were on a C57BL/6 background. 490 ng/min/kg angiotensin II was infused and blood pressure was measured both invasively and noninvasively, as previously described ( ). Animals were maintained in a sterile environment and were regularly screened for infections. For adoptive transfer, mice were anesthetized with xylazine/ketamine and cells were injected via tail vein. Angiotensin II infusion and blood pressure monitoring was begun 3 wk after adoptive transfer. In some experiments, 8 mg/kg etanercept (AmGen) or a neutralizing IFNγ antibody (eBioscience; cl...
Flow cytometry.
Spleens were removed and tissue-disrupted using forceps to release a single-cell suspension, which was passed through a 70-µm sterile strainer. Total blood leukocytes were isolated from the whole heparinized blood after osmotic lysis of excess red blood cells. Cells were centrifuged (800 g ), washed twice with PBS and 0.5% BSA (FACS buffer), counted, resuspended in 1% BSA/PBS, and stored on ice for <30 min. Within 30 min, 10 6 cells were stained for 15 min at 4°C with antibodies and washed twice with FACS buffer. Antibodies used for staining, and in different multicolor combinations, are as follows: FITC anti-CD45 (30-F11); PerCP anti-CD45 (30-F11); APC anti-CD19 (1D3); PE anti-CD4 (GK1.5); APC anti-CD4 (GK1.5); FITC anti-CD4 (GK1.5); PerCP anti-CD8 (53-6.7); APC anti-CD3 (145-2C11); PE anti-I-Ab (AF6-120.1); FITC anti-I-Ab (AF6-120.1); APC anti-CD11b (HL3); APC CD11b...
Analysis of leukocytes in vessels.
Mouse aortas were digested using collagenase type IX (125 U/ml), collagenase type IS (450 U/ml), and hyaluronidase IS (60 U/ml) dissolved in 20 mM Hepes-PBS buffer containing calcium and magnesium for 30 min at 37°C, with constant agitation. Aortas were then passed through a 70-µm sterile cell strainer (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were washed twice with 1% BSA PBS buffer and additionally incubated for 30 min in 37°C with complete media (RPMI; 10%FCS), then washed again, counted, and stained, using multicolor flow cytometry as described in the previous paragraph. An initial gate was applied to exclude cell debris from further analysis ( ), and CD45 staining was used to identify leukocytes within the aortic cell suspension. Within the CD45 + gate, T cells were identified with anti-CD3, -CD4, and -CD8 antibodies, as well as antibodies t...
Immunohistochemistry and immunofluorescence.
For immunohistochemistry, mice were initially perfused with saline, and then with 10% formalin. After tissue processing, the aorta was embedded in paraffin. 5-µm sections of the aorta were prepared from the thoracic aorta segments 1 mm above the diaphragm. After deparafinization, antigen retrieval was performed in citrate buffer, pH 6.0. Blocking was sequentially performed with 2% BSA, normal horse serum (Vector Laboratories), with avidin-biotin block (SP-2001; Vector Laboratories) and 1.5% hydrogen peroxide to remove endogenous peroxidase activity. Staining was performed using a rabbit polyclonal anti-CD3 zeta (Abcam; 1:100 dilution) as a primary antibody for 1 h at room temperature. After this, the sections were extensively washed and incubated for 45 min with a biotinylated secondary antibody from the ABC Vectastain system (PK-6101; Vector). Brown staining was visualized using...
Vascular wall morphometry.
Aortic segments were obtained as described in Immunohistochemistry and immunofluorescence, and sections from 1 mm above the diaphragm were stained with hematoxylin and eosin and were analyzed using an Axioskop with an AxioCam system and AxioVision 4.6 software (all from Carl Zeiss MicroImaging, Inc.). Wall thickness was measured from the internal to the external elastic lamina at 10 evenly spaced sites around the aorta. To determine total cross-sectional wall area, the internal and external elastic laminas were planimetered and their areas subtracted. This difference was reported as cross-sectional wall area.
In vitro effect of angiotensin II on T cell activation.
Spleen-derived T cells isolated by magnetic sorting as described above were suspended in RPMI 1640 with 25 mM Hepes, 1% FBS, 0.1% l -glutamine, and 0.1% penicillin/streptomycin, with or without 100 µM angiotensin II. Cells were plated at a density of 2 × 10 5 per well in 96-well plates precoated with anti-CD3 (BD Biosciences) and cultured for 3 h at 37°C with 5% CO 2. After incubation, cells were collected, washed with 1% BSA/PBS, and stained using anti-CD69-FITC, anti-CD4-APC, and anti-CD8-PerCP. FACS analysis was then performed using an LSR II (BD Biosciences) and FlowJo Software (Tree Star, Inc.).
In vitro effect of angiotensin II on T cell activation.
To analyze the effect of angiotensin II on cytokine production by isolated T lymphocytes, cells were plated at a density of 2 × 10 5 per well in 96-well plates precoated with anti-CD3 and cultured for 48 h at 37°C with 5% CO 2. After the incubation period, the media were collected and analyzed using the Cytometric Bead Array (BD Biosciences) to measure secreted TNF-α.
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Evidence quotes (8)
Role of cytokines in blood pressure regulation and vascular O 2 ·- production. (A) Production of TNF-α (top) and IFN-γ (bottom) measured by cytometric bead array from anti-CD3-stimulated peripheral blood T cells isolated from C57BL/6 and p47 phox-/- mice infused with saline (sham) or angiotensin II for 14 d ( n = 8 in each group). (B) Anti-CD3-stimulated production of TNF-α from spleen-derived T cells in response to in vitro coincubation without (vehicle; n = 6) or with 100 nm angiotensin II (Ang II; n = 6). (C) Noninvasive blood pressure measurements at baseline and during angiotensin II infusion measured by tail cuff in mice injected IP with control IgG ( n = 4) or with anti-TNFα therapy (etanercept, ETA; 8 mg/kg; n = 6) 3 d before and every 3 d throughout the experiment. (D) Aortic O 2 ·- levels after angiotensin II infusion in control mice or mice injected with etanercept ( n = 6) compared with sham-infused mice. In preliminary experiments, we showed that ETA did not directly inhibit contraction of vascular rings in response to angiotensin II. *, P < 0.05 vs. sham; ¶, P < 0.05 vs. α-CD3 + vehicle usi...
C57BL/6, RAG-1 -/-, and AT1a -/- mice were obtained from Jackson ImmunoResearch Laboratories. The p47 phox -/- mice and their appropriate controls were obtained from Taconic. All experimental protocols were approved by the institutional Animal Care and Use Committee at Emory University. All mice were on a C57BL/6 background. 490 ng/min/kg angiotensin II was infused and blood pressure was measured both invasively and noninvasively, as previously described ( ). Animals were maintained in a sterile environment and were regularly screened for infections. For adoptive transfer, mice were anesthetized with xylazine/ketamine and cells were injected via tail vein. Angiotensin II infusion and blood pressure monitoring was begun 3 wk after adoptive transfer. In some experiments, 8 mg/kg etanercept (AmGen) or a neutralizing IFNγ antibody (eBioscience; clone R4-6A2; 0.5 mg per injection per 30 g mouse) was administered i.p. 3 d before and every 3 d during angiotensin II infusion. In some mice, DOCA-salt hypertension was created as previously described, and studies were performed after 40 d from the induction of hypertension ( ).
Spleens were removed and tissue-disrupted using forceps to release a single-cell suspension, which was passed through a 70-µm sterile strainer. Total blood leukocytes were isolated from the whole heparinized blood after osmotic lysis of excess red blood cells. Cells were centrifuged (800 g ), washed twice with PBS and 0.5% BSA (FACS buffer), counted, resuspended in 1% BSA/PBS, and stored on ice for <30 min. Within 30 min, 10 6 cells were stained for 15 min at 4°C with antibodies and washed twice with FACS buffer. Antibodies used for staining, and in different multicolor combinations, are as follows: FITC anti-CD45 (30-F11); PerCP anti-CD45 (30-F11); APC anti-CD19 (1D3); PE anti-CD4 (GK1.5); APC anti-CD4 (GK1.5); FITC anti-CD4 (GK1.5); PerCP anti-CD8 (53-6.7); APC anti-CD3 (145-2C11); PE anti-I-Ab (AF6-120.1); FITC anti-I-Ab (AF6-120.1); APC anti-CD11b (HL3); APC CD11b (M1/70); PE 62L (MEL-14); PE CD25 (PC61); APC CD4 (RM4-5); PerCP CD4 (RM4-5); PE CCR5; FITC γ/δ (GL3); FITC Vβ7; FITC CD44 (IM7); FITC CD69 (H1.2F3); PE TcR β chain (H57-597); PE CD19 (1D3); and PE NK1.1 (PK136).
Mouse aortas were digested using collagenase type IX (125 U/ml), collagenase type IS (450 U/ml), and hyaluronidase IS (60 U/ml) dissolved in 20 mM Hepes-PBS buffer containing calcium and magnesium for 30 min at 37°C, with constant agitation. Aortas were then passed through a 70-µm sterile cell strainer (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were washed twice with 1% BSA PBS buffer and additionally incubated for 30 min in 37°C with complete media (RPMI; 10%FCS), then washed again, counted, and stained, using multicolor flow cytometry as described in the previous paragraph. An initial gate was applied to exclude cell debris from further analysis ( ), and CD45 staining was used to identify leukocytes within the aortic cell suspension. Within the CD45 + gate, T cells were identified with anti-CD3, -CD4, and -CD8 antibodies, as well as antibodies to detect other supplementary surface molecules.
For immunohistochemistry, mice were initially perfused with saline, and then with 10% formalin. After tissue processing, the aorta was embedded in paraffin. 5-µm sections of the aorta were prepared from the thoracic aorta segments 1 mm above the diaphragm. After deparafinization, antigen retrieval was performed in citrate buffer, pH 6.0. Blocking was sequentially performed with 2% BSA, normal horse serum (Vector Laboratories), with avidin-biotin block (SP-2001; Vector Laboratories) and 1.5% hydrogen peroxide to remove endogenous peroxidase activity. Staining was performed using a rabbit polyclonal anti-CD3 zeta (Abcam; 1:100 dilution) as a primary antibody for 1 h at room temperature. After this, the sections were extensively washed and incubated for 45 min with a biotinylated secondary antibody from the ABC Vectastain system (PK-6101; Vector). Brown staining was visualized using 3,3′ diamino-benzidine tetrachloride (peroxidase substrate kit; SK-4100; Vector Labs). Immunofluorescence was performed on frozen 6-µm sections of the same region of the aorta using rat monoclonal anti-TCR (β chain) antibody (H57-597; BD Biosciences; 1:100). A PE-conjugated antibody...
Aortic segments were obtained as described in Immunohistochemistry and immunofluorescence, and sections from 1 mm above the diaphragm were stained with hematoxylin and eosin and were analyzed using an Axioskop with an AxioCam system and AxioVision 4.6 software (all from Carl Zeiss MicroImaging, Inc.). Wall thickness was measured from the internal to the external elastic lamina at 10 evenly spaced sites around the aorta. To determine total cross-sectional wall area, the internal and external elastic laminas were planimetered and their areas subtracted. This difference was reported as cross-sectional wall area.
Spleen-derived T cells isolated by magnetic sorting as described above were suspended in RPMI 1640 with 25 mM Hepes, 1% FBS, 0.1% l -glutamine, and 0.1% penicillin/streptomycin, with or without 100 µM angiotensin II. Cells were plated at a density of 2 × 10 5 per well in 96-well plates precoated with anti-CD3 (BD Biosciences) and cultured for 3 h at 37°C with 5% CO 2. After incubation, cells were collected, washed with 1% BSA/PBS, and stained using anti-CD69-FITC, anti-CD4-APC, and anti-CD8-PerCP. FACS analysis was then performed using an LSR II (BD Biosciences) and FlowJo Software (Tree Star, Inc.).
To analyze the effect of angiotensin II on cytokine production by isolated T lymphocytes, cells were plated at a density of 2 × 10 5 per well in 96-well plates precoated with anti-CD3 and cultured for 48 h at 37°C with 5% CO 2. After the incubation period, the media were collected and analyzed using the Cytometric Bead Array (BD Biosciences) to measure secreted TNF-α.
Machine-readable layer
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"text": "Role of cytokines in blood pressure regulation and vascular O 2 ·- production. (A) Production of TNF-α (top) and IFN-γ (bottom) measured by cytometric bead array from anti-CD3-stimulated peripheral blood T cells isolated from C57BL/6 and p47 phox-/- mice infused with saline (sham) or angiotensin II for 14 d ( n = 8 in each group). (B) Anti-CD3-stimulated production of TNF-α from spleen-derived T cells in response to in vitro coincubation without (vehicle; n = 6) or with 100 nm angiotensin II (Ang II; n = 6). (C) Noninvasive blood pressure measurements at baseline and during angiotensin II infusion measured by tail cuff in mice injected IP with control IgG ( n = 4) or with anti-TNFα therapy (etanercept, ETA; 8 mg/kg; n = 6) 3 d before and every 3 d throughout the experiment. (D) Aortic O 2 ·- levels after angiotensin II..."
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"text": "C57BL/6, RAG-1 -/-, and AT1a -/- mice were obtained from Jackson ImmunoResearch Laboratories. The p47 phox -/- mice and their appropriate controls were obtained from Taconic. All experimental protocols were approved by the institutional Animal Care and Use Committee at Emory University. All mice were on a C57BL/6 background. 490 ng/min/kg angiotensin II was infused and blood pressure was measured both invasively and noninvasively, as previously described ( ). Animals were maintained in a sterile environment and were regularly screened for infections. For adoptive transfer, mice were anesthetized with xylazine/ketamine and cells were injected via tail vein. Angiotensin II infusion and blood pressure monitoring was begun 3 wk after adoptive transfer. In some experiments, 8 mg/kg etanercept (AmGen) or a neutralizing IFNγ antibody (eBioscience; cl..."
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"text": "Spleens were removed and tissue-disrupted using forceps to release a single-cell suspension, which was passed through a 70-µm sterile strainer. Total blood leukocytes were isolated from the whole heparinized blood after osmotic lysis of excess red blood cells. Cells were centrifuged (800 g ), washed twice with PBS and 0.5% BSA (FACS buffer), counted, resuspended in 1% BSA/PBS, and stored on ice for <30 min. Within 30 min, 10 6 cells were stained for 15 min at 4°C with antibodies and washed twice with FACS buffer. Antibodies used for staining, and in different multicolor combinations, are as follows: FITC anti-CD45 (30-F11); PerCP anti-CD45 (30-F11); APC anti-CD19 (1D3); PE anti-CD4 (GK1.5); APC anti-CD4 (GK1.5); FITC anti-CD4 (GK1.5); PerCP anti-CD8 (53-6.7); APC anti-CD3 (145-2C11); PE anti-I-Ab (AF6-120.1); FITC anti-I-Ab (AF6-120.1); APC anti-CD11b (HL3); APC CD11b..."
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"text": "Mouse aortas were digested using collagenase type IX (125 U/ml), collagenase type IS (450 U/ml), and hyaluronidase IS (60 U/ml) dissolved in 20 mM Hepes-PBS buffer containing calcium and magnesium for 30 min at 37°C, with constant agitation. Aortas were then passed through a 70-µm sterile cell strainer (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were washed twice with 1% BSA PBS buffer and additionally incubated for 30 min in 37°C with complete media (RPMI; 10%FCS), then washed again, counted, and stained, using multicolor flow cytometry as described in the previous paragraph. An initial gate was applied to exclude cell debris from further analysis ( ), and CD45 staining was used to identify leukocytes within the aortic cell suspension. Within the CD45 + gate, T cells were identified with anti-CD3, -CD4, and -CD8 antibodies, as well as antibodies t..."
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"name": "In vitro effect of angiotensin II on T cell activation.",
"text": "Spleen-derived T cells isolated by magnetic sorting as described above were suspended in RPMI 1640 with 25 mM Hepes, 1% FBS, 0.1% l -glutamine, and 0.1% penicillin/streptomycin, with or without 100 µM angiotensin II. Cells were plated at a density of 2 × 10 5 per well in 96-well plates precoated with anti-CD3 (BD Biosciences) and cultured for 3 h at 37°C with 5% CO 2. After incubation, cells were collected, washed with 1% BSA/PBS, and stained using anti-CD69-FITC, anti-CD4-APC, and anti-CD8-PerCP. FACS analysis was then performed using an LSR II (BD Biosciences) and FlowJo Software (Tree Star, Inc.)."
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