Induction of chemokine expression by adiponectin in vitro is isoform dependent

Abstract

Adiponectin is reported to have both proinflammatory and anti-inflammatory effects. Because adiponectin circulates in isoforms of various sizes and some responses to adiponectin are isoform dependent, it was postulated that the proinflammatory effects of adiponectin may be isoform specific. To test this theory, peripheral blood mononuclear cells (PBMCs), microvascular endothelial cells (MVECs), and human glomerular mesangial cells (HMCs) were treated with high-molecular-weight (HMW) or low-molecular-weight (LMW) recombinant human adiponectin, and chemokine production was measured. The PBMCs were isolated from healthy volunteers by density gradient centrifugation of ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood through endotoxin-free Ficoll (General Electric Healthcare Bio-Sciences, Uppsala, Sweden). The MVECs were of dermal origin, and the HMCs were isolated from kidneys not suitable for transplantation. Overnight (16 h) incubation with HMW adiponectin (0.01-1 microg/mL for PBMCs; 5-20 microg/mL for MVECs and HMCs) induced a dose-dependent increase in production of monocyte chemoattractant protein-1 and interleukin-8 by PBMCs and MVECs, but it had no effect on HMC chemokine production (n=3-5). LMW adiponectin at the same concentrations did not induce chemokine production in any of the cell types tested, and it did not block cytokine-induced chemokine production by PBMCs or MVECs (n=3-5). These in vitro data suggested that the HMW adiponectin isoform is proinflammatory. To examine the possibility of a relationship between HMW adiponectin and inflammation in vivo, the urine of patients with systemic lupus erythematosus (SLE) and kidney involvement, which was shown previously to contain immunoreactive adiponectin, was examined for the presence of specific adiponectin isoforms by nondenaturing gel electrophoresis. HMW adiponectin was found in the urine of patients with active lupus nephritis. Therefore, HMW adiponectin may contribute to the renal inflammation of SLE.

Figures

Figure 1. Chemokine production by PBMC and MVEC in response to different isoforms of adiponectin

PBMC (a, b) or MVEC (c, d) were untreated (control), or treated with the indicated concentrations of HMW or LMW (tri) adiponectin for 16 hours. Supernatants were harvested and supernatant concentrations of MCP-1 or IL-8 were measured by specific ELISA. Each figure is representative of 3–5 independent experiments done in triplicate. Using PBMC, HMW adiponectin significantly increased MCP-1 and IL-8 compared to control at concentrations of 0.1 and 1μg/ml (*P

Figure 1. Chemokine production by PBMC and MVEC in response to different isoforms of adiponectin

PBMC (a, b) or MVEC (c, d) were untreated (control), or treated with the indicated concentrations of HMW or LMW (tri) adiponectin for 16 hours. Supernatants were harvested and supernatant concentrations of MCP-1 or IL-8 were measured by specific ELISA. Each figure is representative of 3–5 independent experiments done in triplicate. Using PBMC, HMW adiponectin significantly increased MCP-1 and IL-8 compared to control at concentrations of 0.1 and 1μg/ml (*P

Figure 1. Chemokine production by PBMC and MVEC in response to different isoforms of adiponectin

PBMC (a, b) or MVEC (c, d) were untreated (control), or treated with the indicated concentrations of HMW or LMW (tri) adiponectin for 16 hours. Supernatants were harvested and supernatant concentrations of MCP-1 or IL-8 were measured by specific ELISA. Each figure is representative of 3–5 independent experiments done in triplicate. Using PBMC, HMW adiponectin significantly increased MCP-1 and IL-8 compared to control at concentrations of 0.1 and 1μg/ml (*P

Figure 1. Chemokine production by PBMC and MVEC in response to different isoforms of adiponectin

PBMC (a, b) or MVEC (c, d) were untreated (control), or treated with the indicated concentrations of HMW or LMW (tri) adiponectin for 16 hours. Supernatants were harvested and supernatant concentrations of MCP-1 or IL-8 were measured by specific ELISA. Each figure is representative of 3–5 independent experiments done in triplicate. Using PBMC, HMW adiponectin significantly increased MCP-1 and IL-8 compared to control at concentrations of 0.1 and 1μg/ml (*P

Figure 2. Adiponectin receptor expression in PBMC, MVEC, and HMC

The left panel demonstrates the presence of adiponectin receptor R1 and R2 mRNA in resting PBMC, HMC (MC), and MVECs (EC) at the expected sizes of 924 and 446 bases respectively, by RT-PCR. MW indicates molecular weight markers in base pairs. The right panel shows the phosphorylation of AMPK after treatment with AICAR (1 mM, positive control), or HMW and LMW adiponectin both at 10 μg/ml for 10 minutes. The blot was reprobed for actin to demonstrate equivalent loading. One of 4 experiments.

Figure 3. Adiponectin isoforms found in the urine and plasma of patients with SLE nephritis

Urine or plasma proteins from 3 healthy individuals and 6 patients with lupus nephritis were separated on non-reducing, non-denaturing gels to preserve native protein structure, and were immunoblotted for adiponectin. The high, middle (hexamer), and low molecular weight adiponectin isoforms are labeled HMW, MMW, LMW, respectively. In panel 1, CON indicates a urine sample from a healthy individual (representative of 3), and AD represents the adiponectin isoforms produced by human fat as a reference. Panel 2 shows urine adiponectin isoforms during a lupus nephritis flare cycle. The samples were from four and two months before flare (PRE), at renal flare (F), and 4 months after flare (POST). Panel 3 shows plasma (P) and urine (U) adiponectin isoforms from two lupus patients during renal flare (patient 1 lanes 1, 2; patient 2 lanes 5, 6). Lanes 3 and 4 are from a third SLE patient 10 months after a renal flare was diagnosed. The patient was improving but still had over 5 grams of urine protein/day. Panel 4 shows the plasma (P) and urine (U) of two patients blotted for adiponectin in the absence and presence of human recombinant adiponectin (rAdip).