Table 1.
Gene and primer information for qRT-PCR.
Figure 1.
Direct contact between splenocytes and adipocytes alters secreted levels of inflammatory cytokines.
Differentiated 3T3-L1 adipocytes (columns 1 and 2) or isolated murine splenocytes (columns 3 and 4) were cultured alone or together with either direct contact (columns 7 and 8) or no contact (cells separated by a 0.4 µm transwell filter system; columns 5 and 6). Cells were additionally incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h. Secreted cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were quantified by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact were calculated using ANOVA and followed by the post-hoc Bonferroni test. A significant effect was accepted when p<0.05.
Figure 2.
Rates of cytokine secretion and overall levels are affected by direct contact between adipocytes and splenocytes.
Differentiated 3T3-L1 adipocytes and isolated murine splenocytes were co-cultured with no contact (separated by a 0.4 µm transwell filter system) (dashed lines) or with direct contact (solid lines) and incubated with LPS (1 µg/mL) for 0, 8, 24 and 48 h. Cytokines, TNFα (A), IL-6 (B) and MCP-1 (C), were measured in culture media following these incubation times by capture ELISA. All experimental points were performed in triplicate.
Figure 3.
Splenocytes and adipocytes differentially express pro-inflammatory markers.
Differentiated 3T3-L1 adipocytes were co-cultured in direct contact with GFP-expressing murine splenocytes and activated by incubation with LPS (1 µg/mL) for 24 h. Cells were sorted for GFP-positive (splenocyte) and negative (adipocyte) cells by FACS. (A) Representative FACS is shown, with GFP-negative cells gated in R1 and GFP-positive cells gated in R2. (B) Quantitative real-time PCR (qRT-PCR) was used to measure adiponectin and F4/80 expression, specific markers for adipocytes and splenocytes, respectively, to confirm efficiency of cell sorting. (C) TNFα, IL-6 and MCP-1 mRNA expression levels were quantified by qRT-PCR in splenocytes and adipocytes following cell sorting to distinguish individual cytokine expression patterns. All qRT-PCR values were normalized to values obtained for 36B4, a ribosomal 60S subunit protein. Experimental points were measured in duplicate (B and C) for calculation of means and standard deviations. In (C), statistical significance of differing secretion levels between splenocytes and adipocytes was determined by an unpaired two-tailed Student's t-test. A significant effect was accepted when p<0.05.
Figure 4.
Cell contact-mediated enhancement of IL-6 and MCP-1 secretion requires TNFα signaling.
(A and B) Differentiated adipocytes or murine splenocytes (black bars, isolated from wild type mice; gray bars, isolated from TNFα -/- mice) were cultured alone (individual culture, columns 1 and 2) or in co-culture with no contact (columns 3 and 4) or direct contact (columns 5 and 6) as in Figure 1. Cells were incubated in the absence (−) or presence (+) of LPS (1 µg/mL) for 24 h as indicated. (C and D) Wild type (WT) or TNFα -/- (TNFα KO) splenocytes were incubated with adipocytes with direct contact in the presence of LPS (1 µg/mL) and co-cultures were supplemented with 0, 300 pg/mL or 10 ng/mL purified murine TNFα as indicated. Secreted IL-6 (A and C) and MCP-1 (B and D) were quantified by capture ELISA. Experimental points were measured in triplicate (A and B) or duplicate (C and D) for calculation of means and standard deviations. For (A and B) comparison between individual cultures, co-cultures with no contact and co-cultures with direct cell-cell contact from WT and TNFα KO were calculated using ANOVA and followed by the post-hoc Bonferroni test. For C and D all experimental points were compared statistically by ANOVA followed by the Bonferroni post-hoc test, and differed significantly (p<0.01). N.S. denotes no statistical difference.
Figure 5.
Effects of paracrine factors and cell contact on IL-10 secretion and expression.
(A) Differentiated 3T3-L1 adipocytes or wild type murine splenocytes were cultured alone (columns 1 and 2 or 3 and 4, respectively) or together with either no contact (columns 5 and 6) or direct contact (columns 7 and 8). Cells were incubated in the absence (-) (columns 1, 3, 5 and 7) or presence (+) (columns 2, 4, 6 and 8) of LPS (1 µg/mL) for 24 h as indicated. Interleukin-10 (IL-10) in culture media was quantified by capture ELISA. (B) Differentiated 3T3-L1 adipocytes were co-cultured with no contact or direct contact with GFP-expressing murine splenocytes as in Figure 3 and activated by incubation with LPS (1 µg/mL) for 24 h. Splenocytes were sorted as GFP-positive cells by FACS and IL-10 mRNA expression was measured by qRT-PCR. qRT-PCR values were normalized to values obtained for 36B4. In (A) experimental points were measured in triplicate for calculation of means and standard deviations. Comparison between all conditions was calculated using ANOVA followed by the post-hoc Bonferroni test. No statistical significance was found between any measured points (p>0.05). For (B), experimental points were performed in duplicate, and a statistical comparison between no contact and direct contact was made using an unpaired two-tailed Student's t-test.
Figure 6.
NF-κB intracellular signaling pathway participates in the paracrine and cell contact-mediated enhancement of IL-6 and MCP-1 secretion.
(A) Isolated splenocytes were incubated with LPS (1 µg/mL) in the absence (-) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of TNFα were measured by capture ELISA. (B and C) Differentiated 3T3-L1 adipocytes were co-cultured with isolated splenocytes with either no contact or direct contact as indicated. Cells were stimulated with LPS (1 µg/mL) in the absence (−) or presence (+) of 2 µM Bay11-7082 for 24 h. Secreted levels of IL-6 (B) or MCP-1 (C) were measured by capture ELISA. All experimental points were measured in triplicate for calculation of means and standard deviations. In (A) an unpaired two-tailed Student's t test was used to compare Bay11-7082 treated and untreated LPS-stimulated splenocytes. Comparisons between co-cultures (B and C) were calculated using ANOVA followed by the post-hoc Bonferroni test.