Adiponectin Produced by Lymphocytes Inhibits Granulopoiesis.

Abstract

Previous studies by our group have shown that normal unstimulated lymphocytes produce a protein which inhibits colony formation of granulopoietic progenitors, but has no effect on erythroid progenitors. Therefore, this inhibitor was initially designated GIA (granulopoietic inhibitory activity). GIA was identified as a glycoprotein of approximately 30 kDa, with a pI of 7.9 - 8.4. Futhermore, we demonstrated that this inhibitor may have physiological significance in that its production is altered in patients with neutropenia. GIA has proved difficult to characterise to date since it is produced in relatively low amounts although it has a high specific biological activity. Adiponectin is an adipokine reported to share many of the inhibitory characteristics of GIA and has been demonstrated to act as a negative regulator of hematopoiesis and immune response. This study aimed to determine whether GIA is adiponectin or if it represents an adiponectin-like molecule. Lymphocyte conditioned medium (LCM) from lymhocytes cultured at 1 X 106 cells/ml in HL-1 minimal medium was used as a source of GIA. Inclusion of LCM as 10% of the top layer of agar in a myeloid colony assay inhibited growth of CFU-GM by 52 ± 11 % (n=3), confirming the presence of the inhibitory activity. RNA and protein from lymphocytes and LCM harvested over a 7 day culture period were subsequently investigated for adiponectin expression. Western blot analysis demonstrated a distinct banding pattern in days 3-7 LCM corresponding to monomers, dimers, trimers and greater. This is consistent with adiponectin which circulates as a multimer of trimers. Characterisation of GIA at the transcript level confirmed that GIA is in fact adiponectin. The N-terminal collagenous domain, C-terminal globular domain and full length adiponectin were amplified by RT-PCR analysis. Adiponectin is thought to be secreted exclusively from adipocytes and much of our current knowledge of this molecule relates to its metabolic functions. Our study provides evidence that adiponectin is also produced by lymphocytes and may play a role in the pathogenesis of neutropenia.