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.

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.

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.

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.