T-cell contact-dependent regulation of CC and CXC chemokine production in monocytes through differential involvement of NFkappaB: implications for rheumatoid arthritis

Abstract

We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFalpha). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFkappaB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IkappaBalpha, the natural inhibitor of NFkappaB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFalpha, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFkappaB-dependent and NFkappaB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.

Figure 1

Activated T cells induce contact-dependent chemokine production by human macrophages. Lymphocytes were left unstimulated or were stimulated with either anti-CD3 for 48 hours (Ttcr cells) or a 'cocktail' of inflammatory cytokines (tumour necrosis factor alpha (TNFα), IL-2, IL-6) (Tck cells) for 8 days, before fixation. The unstimulated, Ttcr and Tck populations were then cultured with macrophage-colony stimulating factor-differentiated monocytes (ratio 7:1) for 18 hours. Culture supernatants were then isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) measured by ELISA. In some cases, a porous membrane insert was used to physically separate the two populations, while allowing the transition of soluble mediators. Results are shown from (a) Ttcr-cell lymphocyte cultures and (b) Tck-cell lymphocyte cultures. Data represent a mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant differences in chemokine detection are indicated.

Figure 2

Differential utilization of NFκB in activated-T-cell contact-dependent chemokine production by human macrophages. Macrophage-colony stimulating factor-differentiated monocytes were infected with AdIκBα or Ad0, an empty control virus. After a further 2 days of culture and replating, anti-CD3-activated T cells (Ttcr cells) and cytokine-activated T cells (Tck cells) were added at a lymphocyte:monocyte ratio of 7:1. After 18 hours, culture supernatants were isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) were measured simultaneously by ELISA. (a) MIP-1α levels in uninfected, Ad0-infected (multiplicity of infection (MOI) 200:1) and AdIκBα-infected (MOI 40:1, 80:1 and 200:1) monocyte cultures when stimulated with Ttcr-cells or Tck-cells. (b) and (c) Levels of CC and CXC chemokines in Ad0-infected and AdIκBα-infected monocytes (MOI 80:1) following stimulation with (b) Ttcr cells and (c) Tck cells. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant reduction in chemokine levels in AdvIκBα-infected (as compared with Ad0-infected) cultures is indicated.

Figure 3

IκBα overexpression significantly inhibits rheumatoid T-cell-induced macrophage chemokine secretion of CXC, but not CC, chemokines. Using anti-CD3 labelled Dynabeads, synovial T cells were enriched from the mixed cell population obtained following enzymatic dissociation of synovial tissue samples from rheumatoid arthritis (RA) patients. Fixed RA T cells were cultured with macrophage-colony stimulating factor-differentiated monocytes infected with Ad0 and AdvIκBα at a T cell:monocyte ratio of 7:1 as described in Figure 2. After 18 hours, culture supernatants were isolated and levels of CC chemokines (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), RANTES) and CXC chemokines (IL-8, growth-related gene product alpha (GROα) and interferon-gamma-inducible protein (IP-10)) were measured by ELISA. (a) Levels of chemokines for monocytes infected with Ad0 and AdIκBα (multiplicity of infection (MOI) 80:1) following stimulation with RA T cells. (b) GROα levels in uninfected, Ad0-infected (MOI 200:1) and AdvIκBα-infected (MOI 20:1, 40, 80:1 and 200:1) monocyte cultures when stimulated with RA T cells. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments. Statistically significant reduction in chemokine levels in AdIκBα-infected (as compared with Ad0-infected) cultures is indicated.

Figure 4

The phosphatidyl-inositol-3-kinase pathway regulates both NFκB-dependent and NFκB-independent contact-dependent chemokine production. Macrophage-colony stimulating factor-differentiated monocytes were preincubated for 30 minutes in the presence or absence of variable amounts of LY294002 (as shown) before being stimulated with anti-CD3-activated T cells (Ttcr) or cytokine-activated T cells (Tck) at a T cell:monocyte ratio of 7:1. After 18 hours, culture supernatants were isolated and levels of (a) macrophage inflammatory protein 1 alpha (MIP-1α) (CC chemokine) and (b) interferon-gamma-inducible protein (IP-10) (CXC chemokine) were measured by ELISA. Data represent the mean of triplicate cultures ± standard deviation and are representative of at least three experiments.