E. Castelli¹, C. Apicella², C. Bartella², G. Innocente², M. Vitiello², F. Mancini², I. Coletta², G. Mangano², L. Durando², A. Guglielmotti²

There is evidence that Monocyte Chemotactic Proteins (MCP-s), and in particular MCP-1/CCL2 and MCP-3/CCL7 play an important role in several pathologies such as inflammation and cancer (Jia et al., 2008; Fioretti et al., 1998). The prevention of inflammation and cancer through blockade of the chemokines/chemokine receptors system is a major target for pharmacological intervention. Several cell types are known to produce MCP-1 following a variety of stimuli, while little is known about MCP-3. Aim of this work was to characterize MCP-3 expression and production in comparison with MCP-1 in various representative cell targets for inflammation and cancer, in order to set-up in vitro models to screen potential MCP-s inhibitors.
Cells, such as MonoMac 6 and THP-1 (human monocytic/macrophage cell lines), normal human mesangial cells (NHMC) and prostate cancer cell lines (PC-3) were exposed to proinflammatory stimuli (LPS, IL-1β, TNF-α) at different concentrations and different incubation times. Chemokine production was evaluated in the supernatant by ELISA. Total RNA extracted from cell pellets was reverse transcribed in cDNA and MCP-s expression was detected by Real Time PCR (Q-PCR). Cell viability was measured to assess toxicity effects. Moreover cells were characterized for CCR2 expression by western blotting technique.
Results obtained confirmed that monocytic/macrophage cell lines are a major source of MCP-1 following stimulation with LPS and TNF-α, alone or in combination with IFNγ. In the same conditions, MCP-3 expression and production was also induced, but at much lesser extent and with a slower onset time. NHMC, both following LPS and IL-1βstimulation, were able to induce production of MCP-1 and, as observed for monocytes, low amount of MCP-3.  PC-3 human prostate cancer cells were able to produce detectable amounts of MCP-1 in response to LPS and IL-1β, while MCP-3 levels were undetected in the condition and at time points used. Analysis of CCR2 expression revealed that MCP-1/MCP-3 receptor is expressed in these cells. Data obtained show that the best experimental model useful to study simultaneously MCP-1 and MCP-3 could be one of monocyte/macrophage origin. Experimental conditions should be refined in order to increase MCP-3 expression and production.
 
Jia et al.(2008). J Immunol. 180(10): 6846–6853.
Fioretti et al. (1998)J Immunol.161: 342–346.