Peptidoglycan and lipoteichoic acid modify monocyte phenotype in human whole blood.

We examined the influence of the gram-positive cell wall products peptidoglycan (PepG) and lipoteichoic acid (LTA), compared to lipopolysaccharide (LPS), on the monocyte expression of receptors involved in antigen presentation (HLA-DR, B7.1, and B7.2), cell adhesion (intercellular adhesion molecule-1 [ICAM-1] and lymphocyte function associated antigen-3 [LFA-3]), phagocytosis (Fc gamma RI), and cell activation (CD14). We also evaluated possible influences of the immunosuppressive drugs cyclosporine A, tacrolimus, and sirolimus on the expression of these receptors. Pretreatment of whole blood for 4 h with the immunosuppressive drugs did not influence the expression of the surface receptors in normal or stimulated blood. Stimulation with both PepG and LTA caused significant up-regulation of the surface expression of ICAM-1 and HLA-DR on whole blood monocytes, similar to that obtained with LPS, whereas B7.1, B7.2, LFA-3, and Fc gamma RI were not modulated. PepG and LTA also caused increased expression of CD14, whereas LPS down-regulated this molecule. In contrast, we did not detect any significant influence of any of the bacterial products on the plasma concentration of soluble CD14. We hypothesized that the increased expression of surface CD14 in blood stimulated with PepG would prime for cellular activation by LPS. Indeed, we show that PepG and the partial PepG structure muramyl dipeptide acted in synergy with LPS to cause the release of tumor necrosis factor-alpha. The results suggest that PepG and LPS provoke partly different responses on monocyte phenotype and that CD14 may play different roles in the innate response to gram-positive and gram-negative bacteria.

Sirolimus interferes with the innate response to bacterial products in human whole blood by attenuation of IL-10 production.

Current immunosuppressive strategies are aimed at abrogating the allospecific T-cell response against donor tissues or organs. However, little information is yet available on the potential influences of these drugs on innate immune responses. In order to address this, we have employed a whole blood model. Human whole blood was pretreated with sirolimus, cyclosporine A or tacrolimus in therapeutic as well as supra therapeutic doses, and subsequently stimulated with lipopolysaccharide (LPS), peptidoglycan (PepG) or lipoteichoic acid (LTA). Plasma cytokine analyses revealed a potent inhibitory effect of sirolimus on interleukin(IL)-10 production induced by all bacterial products tested. In contrast, cyclosporine A and tacrolimus inhibited the tumour necrosis factor (TNF)-alpha production in response to LPS, but not to PepG and LTA. Using a quantitative mRNA analyses, we also observed that sirolimus significantly decreased the IL-10 mRNA accumulation to sub-basal levels in peripheral blood mononuclear cells (PBMC). This suggests that the sirolimus inhibits IL-10 production by interfering with the IL-10 gene transcription. However, the molecular mechanism of this inhibition remains unclear. Based on the present study and observations by others, we postulate that the clinical use of the sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.

Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model.

We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated from Staphylococcus aureus to induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-alpha and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-alpha, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-alpha secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-alpha, IL-6, and IL-10. The TNF-alpha mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-alpha but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.