IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation -- McLoughlin et al. 102 (27): 9589 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

McLoughlin et al. 10.1073/pnas.0501794102.

Supporting Information

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Supporting Figure 7
Supporting Table 1
Supporting Table 2
Supporting Figure 8
Supporting Table 3

Supporting Figure 7

Fig. 7. Isotype control staining to complement Fig. 1C. FACS analysis was performed as outlined in Fig. 1C. Data shows representative scatter plots. Data for CD3 and B220 staining is as seen for the wild-type data in Fig. 1C and is shown for comparison. The scatter plot for isotype control (IC) antibody (IgG2a) staining is depicted.

Supporting Figure 8 Fig. 8.
Densitometry evaluation to complement Fig. 6C. (A) The relative banding intensity of extracellular signal-regulated kinase (ERK)1/2 bands for each time point was quantified by using image j. Analysis showed that the mean staining intensity was 17,384 ± 3,753 (horizontal line and the filled box). (B) The relative proportion of STAT3 in each mouse strain is shown as a ratio to ERK1/2, which acts as a loading control. The values presented in boxes shows the mean ratio ± SD for each of the strains. Levels of STAT3 were significantly reduced only in the compound gp130^Y757F:Y757FSTAT3^+/– mice (P < 0.05).

Table 1. Phenotypic characteristics of T cells from WT and IL-6^–/– mice

% marker expression	Peripheral Blood		Spleen		Peritoneal cavity		Bone marrow
% marker expression	WT	IL-6^–/–	WT	IL-6^–/–	WT	IL-6^–/–	WT	IL-6^–/–
CD3⁺	14.4 ± 2.4	16.4 ± 1.5	42.7 ± 7.1	35.5 ± 6.4	10.1 ± 0.9	9.58 ± 1.0	4.3 ± 0.2	4.3 ± 0.4
CD4⁺	8.8 ± 0.8	10.9 ± 2.7	22.5 ± 3.0	18.3 ± 2.8	6.7 ± 0.8	3.0 ± 1.1*	5.2 ± 0.1	4.4 ± 0.7
CD8⁺	7.7 ± 0.6	8.0 ± 0.9	16.8 ± 2.2	14.8 ± 2.2	8.8 ± 2.3	7.2 ± 1.8	9.9 ± 1.1	6.8 ± 1.3
CD3⁺/CD25⁺	4.2 ± 0.8	2.8 ± 0.4	8.1 ± 1.6	6.0 ± 1.8	5.6 ± 0.5	5.9 ± 0.7	3.9 ± 0.5	3.9 ± 0.4
CD3⁺/CD28⁺	12.0 ± 2.2	9.5 ± 2.0	17.3 ± 1.5	14.7 ± 1.4	11.8 ± 0.8	7.7 ± 0.8	6.6 ± 1.3	5.2 ± 0.8
CD3⁺/CD62L⁺	9.3 ± 1.8	10.7 ± 2.4	14.2 ± 2.8	15.3 ± 2.5	9.7 ± 2.1	5.6 ± 0.9	6.9 ± 0.9	5.6 ± 0.4
CD3⁺/CCR3⁺	6.0 ± 1.5	2.9 ± 0.1	9.6 ± 1.9	7.0 ± 2.6	3.4 ± 0.5	4.1 ± 0.9	5.7 ± 0.7	5.0 ± 0.8
CD3⁺/CCR5⁺	5.8 ± 0.8	4.5 ± 0.3	6.3 ± 1.2	6.5 ± 1.3	5.3 ± 1.4	4.0 ± 1.0	3.3 ± 0.3	3.0 ± 0.3
CD3⁺/CXCR3⁺	6.0 ± 0.8	5.1 ± 0.4	10.0 ± 2.0	7.5 ± 1.0	1.3 ± 0.4	1.5 ± 0.3	3.8 ± 0.5	2.6 ± 0.5

T cells from nonchallenged WT and IL-6^–/– mice were analyzed by flow cytometry as indicated. The proportion of single- or dual-positive populations is expressed as the mean percentage ± SEM (n = 5 mice per determination; *, P < 0.05).

Table 2. Expression of T cell activation markers after Staphylococcus epidermidis
treatment

% Marker Expression	Peripheral blood		Peritoneal cavity
% Marker Expression	WT	IL-6^–/–	WT	IL-6^–/–
CD3⁺	18.9 ± 1.0	20.4 ± 1.8	42.9 ± 4.8	27.3 ± 4.2*
CD3⁺/CD25⁺	2.4 ± 0.2	2.9 ± 0.2	5.8 ± 0.7	4.1 ± 0.8
CD3⁺/CD28⁺	10.2 ± 0.6	13.8 ± 0.8	9.1 ± 1.6	7.9 ± 0.8

Staphylococcus
epidermidis-induced peritoneal inflammation was established in WT and IL-6^–/– mice. At 48 h, leukocytes were isolated from the peritoneal cavity and peripheral blood, and expression of T cell activation markers on the CD3⁺ population were analyzed by flow cytometry. Values represent the mean ± SEM (n = 5 mice per group, *, P < 0.05).

Table 3. CCR3 and CCR5 expression on gp130 knock-in mice

Strains	CD3⁺CCR3⁺	CD3⁺CCR5⁺
WT	6.0 ± 1.1	9.9 ± 1.3
gp130^Y757F:Y757F	2.8 ± 0.3	4.5 ± 0.1
gp130^Y757F:Y757FSTAT3^+/–	2.3 ± 0.8	3.9 ± 0.8
gp130D^STAT:DSTAT	5.8 ± 0.0	8.3 ± 0.0

Circulating T cells from nonchallenged WT and gp130 knock-in mice were analyzed by flow cytometry. The proportion of CD3⁺CCR3⁺ and CD3⁺CCR5⁺ dual-positive populations is expressed as the mean percentage ± SEM (n = 2-3 mice per determination).