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Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF stimulate the differentiation of rat alveolar macrophages (AM) into multinucleated giant cells (MGC) with distinct phenotypes (type 1 and type 2 MGC). In the present study, we analyzed the profile of cytokine gene expression induced respectively, by M-CSF and GM-CSF during rat AM differentiation using reverse transcription-PCR. Enhanced mRNA expression for IL-1alpha, TNF-alpha, and TGF-beta1 was observed 3 h after treatment with M-CSF (50 U/ml) or GM-CSF (50 U/ml). In contrast, IL-6 mRNA expression was increased by GM-CSF but not M-CSF. Kinetic analysis indicated that GM-CSF stimulated IL-6 expression early (1.5 h), with maximal effect observed at 24 h and up to 5 days thereafter. Increased mRNA levels for IL-6 were associated with higher IL-6 activity in the culture media of differentiating AM. IL-6 activity was stimulated 3 h after treatment with GM-CSF and increased with time (up to 5 days). Interestingly, addition of exogenous IL-6 (20-100 ng/ml) alone or in combination with GM-CSF to AM cultures increased slightly and selectively the formation of MGC with type 2 phenotype. Conversely, neutralization of endogenous IL-6 during AM differentiation into MGC inhibited significantly (up to 50%) the formation of type 2 MGC. These results suggest a role for IL-6 in the formation of type 2 MGC and provide some insights into the mechanisms of MGC formation and the processes that regulate it positively.

The efficacy of all-trans retinoic acid (RA) in the treatment of acute promyelocytic leukemia results from the ability of RA to differentiate these peculiar leukemic cells. The efficacy of differentiation therapy could be improved and extended to other forms of leukemia by associating retinoids with other differentiating agents. Here we have compared the effects of different combinations of retinoids with 1 alpha,25-dihydroxyvitamin D3 (VD3) analogs on myelomonocytic cell lines HL-60, U937 and THP-1. All-trans RA, its natural isomer 9-cis RA and the arotinoid TTNPB, which differ by their respective specificities for the RA receptor families (retinoic acid receptor and retinoid X receptor), were found to cooperate with VD3 in inhibiting cell growth of the leukemic cell lines. Although the three cell lines displayed different susceptibilities to retinoids, each molecule was able to cooperate with VD3 in inducing U937 cell differentiation. Because the effects of VD3 on calcium metabolism limit its therapeutic use, we studied the effects of two synthetic analogs, MC903 and KH1060. Both agents cooperate with RA, acting more efficiently than the natural molecule in inhibiting cell growth and inducing some parameters of U937 cell differentiation. These results extend our previous data demonstrating that RA and VD3 exert synergistic effects on the differentiation of the myelomonocytic cell line U937. They demonstrate that combinations of agents able to inhibit leukemia cell growth with limited side effects may be found among a wide array of retinoids and vitamin D3 analogs.

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages.

Homoretinoic and bishomoretinoic acid have been synthesized. Fast reaction under ultrasonic activation (Wolff rearrangement, saponification) produced compounds in high yields. Only the bishomo analogue exhibits a significative activity alone and a synergistic effect with vitamin D3 on the differentiation of U937 leukemic cell line.