Extensive studies in the past 10 years have suggested that heat shock protein 60 (Hsp60) and Hsp70 may be potent activators of the innate immune system capable of inducing pro-inflammatory cytokine production by macrophages. However, we have recently demonstrated that the reported pro-inflammatory cytokine-inducing effect of Hsp60 and Hsp70 was due to contaminating lipopolysaccharide (LPS) and LPS-associated molecules. In the current study, we determined whether highly purified, essentially LPS-free recombinant human Hsp60 (rhHsp60) and rhHsp70 had any cytokine-inducing effect. Using gene expression array, we demonstrated that at 2 and 4 It after treatment, while LPS (1 ng/ml) markedly enhanced the expression of a number of cytokine genes, rhHsp60 and rhHsp70 (5 mug/ml) had no effect on any of the 96 common cytokine genes. Reverse transcriptase-polymerase chain reaction analysis and enzyme-linked immunosorbent assay of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) supported the above observation. These data suggest that rhHsp60 and rhHsp70 do not activate cytokine genes in murine macrophages. (C) 2004 Elsevier Inc. All rights reserved.