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RANKL expression in human T-lymphocytes requires cooperative signaling through the T-cell receptor and adhesion molecule CD2

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Introduction

T-lymphocytes contribute to osteolysis in rheumatic diseases through their production of the osteoclastogenic cytokine RANKL. Mitogenic stimulation of lymphocytes has been shown to induce expression of RANKL; however, the extracellular events leading to its production have not been identified.

Aim

We sought to determine whether cell-to-cell interactions through adhesion molecules such as lymphocyte function-associated antigen 2 (CD2) were necessary to promote RANKL secretion from T-cells.

Patients and methods

Human CD4+ and CD8+ T cells were purified by negative selection. PBMC from rheumatoid arthritis (RA) patients were obtained from Conversant Healthcare Systems. Cells were cultured in medium supplemented with human serum, IL-2, IL-7, M-CSF and 1alpha,25-dihydroxyvitamin D3 in the presence of various combinations of bead bound anti-CD3, anti-CD2 and anti-CD28 antibodies for 4 days. Total RANKL was determined by osteoprotegrin capture sandwich ELISA and secreted cytokines by Meso Scale Discovery assay. T-cell activation was determined by flow cytometry using CD69 and CD25.

Results

RANKL secretion by healthy donor PBMC was first detected after 72 hr incubation with anti-CD3/CD2/CD28 antibodies. Anti-CD3/CD28 antibodies failed to induce detectable levels of RANKL. Both CD4+ and CD8+ T-cells produced RANKL only in response to the combined cross-linking of CD3 and CD2 whereas cross-linking of CD3 and CD28 was insufficient to promote RANKL expression even though the T-cells were activated. Those conditions (anti-CD3/CD2) that led to increased RANKL secretion induced significantly lower levels of TNF-α and IL-4 as compared to the combinations of anti-CD3/CD28 or anti-CD3/CD2/CD28 antibodies. Anti-CD2/CD28 antibodies did not induce RANKL secretion. PBMC from RA patients also secreted RANKL in a CD3/CD2-dependent manner and at levels similar to PBMC from healthy donors.

Conclusions

Our results demonstrate that T-lymphocytes can generate RANKL following the co-ligation of the TCR/CD3 and the adhesion molecule CD2 in the absence of the co-stimulatory receptor CD28, suggesting that interactions between T-cells and non-traditional APCs (e.g., synovial fibroblasts) could lead to the production of osteoclastogenic cytokines without the need for other co-stimulatory signals.

Author information

Correspondence to B P Harvey.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Harvey, B.P., Kaymakcalan, Z. RANKL expression in human T-lymphocytes requires cooperative signaling through the T-cell receptor and adhesion molecule CD2. J Transl Med 8, O4 (2010) doi:10.1186/1479-5876-8-S1-O4

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Keywords

  • Rheumatoid Arthritis
  • Synovial Fibroblast
  • CD28 Antibody
  • RANKL Expression
  • Meso Scale Discovery