CCL19 and CCL21 modulate the inflammatory milieu in atherosclerotic lesions

Figures & data

Table 1 Primer sequences, including forward (FW) and reverse (RV) sequences and optimal temperatures used for quantitative real-time polymerase chain reaction

mRNA		Oligonucleotide sequence (5′→3′)	Annealing temperature (°C)
Actb	FW	GACGGCCAGGTCATCACTATTG	57
	RV	CCACAGGATTCCATACCCAAGA
Ccl19	FW	TGTGTTCACCACACTAAGGGG	58
	RV	CCTTTGTTCTTGGCAGAAGACT
Ccl21-Ser	FW	ATCCCGGCAATCCTGTTCTC	58
	RV	GGGGCTTTGTTTCCCTGGG
Ccl21-Leu	FW	GTGATGGAGGGGGTCAGGA	58
	RV	GGGATGGGACAGCCTAAACT
Ccr7	FW	CAGGTGTGCTTCTGCCAAGAT	58
	RV	GGTAGGTATCCGTCATGGTCT
Cd3e	FW	ATGCGGTGGAACACTTTCTGG	58
	RV	GCACGTCAACTCTACACTGGT
Tnfa	FW	CCCTCACACTCAGATCATCTTCT	58
	RV	TGCTACGACGTGGGCTACAG
Ifng	FW	ATGAACGCTACACACTGCATC	56
	RV	CCATCCTTTTGCCAGTTCCTC
Cc12	FW	TTAAAAACCTGGATCGGAACCAA	55
	RV	GCATTAGCTTCAGATTTACGGGT
Mmp13	FW	ACCTCCACAGTTGACAGGCT	58
	RV	AGGCACTCCACATCTTGGTTT
Tf	FW	CCTGGGCCTATGAAGCAAAG	57
	RV	GTTGGTCTCCGTCTCCATGAA

Table 2 Blood and baseline parametersa

Parameter	plt/plt/Ldlr ^−/− (n=9) ± SD	Control (n=9) ± SD	P-value
Leukocytes (/nL)	2.50±0.91	4.18±2.33	NS
Erythrocytes (/pL)	4.23±2.02	5.23±2.69	NS
Hemoglobin (g/dL)	6.34±3.55	8.78±3.70	NS
Thrombocytes (/nL)	206.33±80.59	322.83±263.59	NS
Triglycerides (mg/dL)	473.3±78.82	768.6±142.50	NS
Total cholesterol (mg/dL)	660.0±216.8	780.88±109.62	NS
LDL cholesterol (mg/dL)	541.0±187.4	648.3±124.9	NS
HDL cholesterol (mg/dL)	22.29±6.89	26.0±7.48	NS
Body weight (g)	27.52±1.45	27.75±2.11	NS
Length (cm)	9.59±0.13	9.60±0.13	NS

Notes:

a Body weight and length, lipid profile, and hematological parameters were measured on the day of tissue harvesting of plt/plt/Ldlr ^−/− mice and controls. All values are shown as means ± SD.

Abbreviations: SD, standard deviation; NS, not significant; LDL, low-density lipoprotein; HDL, high-density lipoprotein.

Table 3 Quantitative real-time polymerase chain-reaction results for different cytokines, chemokines, and transcription factors from the thoracic aortaa

Target gene	plt/plt/Ldlr ^−/− (n=9) ΔCT ± SD	Control (n=9) ΔCT ± SD	P-value
Ccl19	192.29±104.86	543.56±368.12	0.0031
Ccl21-Ser	64.43±18.64	219.90±92.19	0.0002
Ccl21-Leu	222.75±144.26	49.94±24.73	0.0002
Ccr7	252.83±76.39	35.51±28.15	0.0001
Cd3e	114.94±121.11	53.63±28.70	0.0056
Tf	64.45±40.92	122.23±58.63	0.0003
Mmp13	113.14±159.15	514.72±231.62	0.0001
Ccl2	149.69±169.92	517.33±178.82	0.0010
Ifng	85.15±143.34	378.58±138.16	0.0001
Tnfa	5.26±5.99	33.51±14.09	0.0001

Notes:

a Values are normalized to β-actin and expressed as cDNA copies/1,000 β-actin copies. All values are shown as means ± SD.

Abbreviations: SD, standard deviation; cDNA, complementary deoxyribonucleic acid.

Figure 1 No changes on plaque burden in plt/plt/Ldlr ^−/− mice.

Notes: Representative photomicrographs of Oil Red O immunostaining from aortic roots of plt/plt/Ldlr ^−/− mice (n=9) and control group (n=9) are shown at 4× magnification (A) Quantitative analysis of lesion area (μm²), fractional stenosis (%), and maximum stenosis (%) (B). Results are presented as box plots displaying means and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers.
Abbreviation: NS, not significant.

Figure 2 Higher plaque stability in plt/plt/Ldlr ^−/− mice.

Notes: Representative photomicrographs of immunohistochemistry stainings of smooth muscle cells (SMCs; α-SM actin, red staining [A, B]) and collagen and elastin (Movat’s staining, [C, D]) from aortic root of plt/plt/Ldlr ^−/− mice (n=9) and control group (n=9). Magnification (10×) of photomicrographs of A and C are shown in B and D. Quantitative analysis of the number of smooth muscle cells in the whole lesion area (E) and in the region of the fibrous cap (F). Collagen and elastin were calculated as percentage of Movat’s⁺-stained plaque area to total lesion area (G + H). The quantitative data are presented as box plots displaying means and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers (E–G).
Abbreviation: NS, not significant.

Figure 3 Increased lesional leukocyte infiltration in plt/plt/Ldlr ^−/− mice.

Notes: Representative photomicrographs of immunohistochemistry stainings of T-cells (CD3; red staining [A]), macrophages (MAC2; brown staining [B]), B cells (B220; red staining [C]) are shown at 10× magnification. Quantitative analysis of T-cells, macrophages, and B cells in plaque of plt/plt/Ldlr^−/− mice (n=9) and control group (n=9) (D). The quantitative data are presented as box plots displaying means and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers.

Table 4 Multiplex ELISA results for different cytokines and chemokines in blood serum of mice

Target protein	plt/plt/Ldlr ^−/− (n=9) pg/mL ± SD	Control (n=9) pg/mL ± SD	P-value
CCL2 (MCP-1)	67.12±11.15	117.1±36.63	0.0111
CCL3	124.2±115.1	46.61±22.74	0.0401
CCL5	210.4±126.9	79.36±42.35	0.0055
TNFα	347.6±67.72	703.6±274.4	0.0115
IFNγ	292.7±37.32	613.7±298	0.0311
IL-1α	220±106	206.5±108.4	NS
IL-1β	95.85±26.75	183.2±75.59	0.0111
IL-2	50.63±12.59	119.3±46.16	0.0115
IL-3	37.19±83.90	83.90±38.55	0.0161
IL-4	175.12±71.09	169.2±65.76	NS
IL-5	42.84±25.62	39.80±19.33	NS
IL-6	369.9±478.8	2539±2175	0.0025
IL-10	85.27±34.63	83.85±45.67	NS
IL-12	123.5±23.59	244.5±112.5	0.0311
IL-17	41.90±5.52	93.71±36.33	0.0464

Note: All values are shown as means ± SD.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; NS, not significant.

Figure 4 CCL19 induces expression of Tnfa and Ifng in murine macrophages. RAW cells (murine macrophage cell line) were stimulated with either 100 ng CCL19 or 100 ng CCL21, and after 2 and 8 hours messenger ribonucleic acid levels of Tnfa (A, C) and Ifng (B, D) were measured by quantitative real-time polymerase chain-reaction analysis. Values are normalized to β-actin and expressed as complementary deoxyribonucleic acid (cDNA) copies/1,000 β-actin copies. Quantitative data of five independent experiments are presented in bar graphs ± standard deviation (A–D).

Note: *P<0.03 versus unstable RAW cells.

Table 5 Cellular composition of peripheral lymph nodes and spleena

Cells of interest	plt/plt/Ldlr ^−/− (n=9) ± SD	Control (n=9) ± SD	P-value
Peripheral lymph nodes
CD3^+	28.78±5.07	31.60±2.74	NS
CD3^+/CD4^+	12.64±1.52	15.62±1.68	NS
CD3^+/CD8^+	6.10±3.89	7.64±5.50	NS
CD3^+/CD4^+/CD8^+	2.32±1.21	2.67±0.81	NS
CD3^+/CD4^+/CD44^+	5.29±1.18	9.15±2.57	0.0163
CD19^+	40.17±5.06	40.72±3.34	NS
CD19^+/CD25^+	4.67±1.69	5.02±2.73	NS
CD11c^+	0.98±0.17	1.03±0.41	NS
CD11c^+/CD83^+	0.21±0.07	0.22±0.09	NS
Spleen	(n=5) ± SD	(n=5) ± SD
CD3^+	35.56±4.96	36.42±5.47	NS
CD3^+/CD4^+	13.48±3.22	13.25±2.39	NS
CD3^+/CD8^+	7.56±2.48	8.15±1.86	NS
CD3^+/CD4^+/CD8^+	4.21±0.87	4.16±0.54	NS
CD3^+/CD4^+/CD44^+	7.36±2.69	8.05±1.85	NS
CD19^+	44.23±7.15	46.89±6.22	NS
CD19^+/CD25^+	6.72±2.43	5.75±2.96	NS
CD11c^+	1.64±0.31	1.25±0.38	NS
CD11c^+/CD83^+	0.24±0.05	0.27±0.06	NS

Notes:

a Flow-cytometry analysis was performed to analyze the amount of T-cells (CD3/CD4/CD8/CD44), B cells (CD19/CD25), dendritic cells (CD11c/CD83), their subtypes, and grade of activation in peripheral lymph nodes and spleen. Values are shown as percentage of positive cells/total leukocytes in lymph node or spleen and as means ± SD.

Abbreviations: SD, standard deviation; NS, not significant.

Figure 5 Lipid-loaded macrophages in atherosclerotic lesions and effects of CCL19 on oxidized low-density lipoprotein (oxLDL) uptake and foam-cell formation. Representative photomicrographs of immunohistochemistry stainings of macrophages (MAC2; brown staining) from aortic roots of plt/plt/Ldlr ^−/− mice (n=9) and control group (n=9) are shown at 4× magnification. Magnification (20×) shown below the images (black arrows point at macrophages) (A). Quantitative analysis of the size of lesional macrophages (μm²) is demonstrated. Results are presented as box plots displaying mean and 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers (B). Effects of CCL19 on foam-cell formation (C, D). Representative Oil Red O staining of human monocyte-derived macrophages are shown. Groups: control (no oxLDL), oxLDL, and oxLDL plus CCL19 (C). Quantitative analysis of the Oil Red O-positive cell area/cell (oxLDL/cell [%]). Data are demonstrated as bar graphs ± standard deviation of five independent experiments (D). Quantitative analysis of five independent fluorescence-activated cell-sorting experiments for CD36 expression on the surface of human monocyte-derived macrophages after stimulation with 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (Dil)-labeled oxLDL or Dil-labeled oxLDL in addition to CCL19 or unstimulated macrophages (control). Data are presented as bar graphs ± standard deviation (E).

Abbreviation: NS, not significant.