Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways

Abstract

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1alpha (IL-1alpha), IL-1ra, and tumor necrosis factor alpha (TNF-alpha) production, whereas blocking of the ERK1/2 pathways inhibited IL-1alpha, IL-1beta, and IL-1ra but not TNF-alpha production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1alpha production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.

FIG. 1

Measurement of mCD14 and intracellular CD14 by fluorescence-quenching assay. mCD14 and intracellular CD14 were detected by staining unstimulated monocytes (left) and LPS-stimulated monocytes (1 μg/ml) (right) with anti-CD14-FITC MAba. Histograms represent cells incubated with (b) or without (c) rabbit anti-fluorescein-Texas red conjugate, fixed, and analyzed by FACS. At least 3,000 CD14⁺ events/sample were acquired for analysis. An isotypic control was used as negative control (a). Results are expressed as log green fluorescence intensity versus number of cells. Results are those from one representative experiment out of three performed with cells from different donors.

FIG. 2

Assessment of CD14 internalization in LPS-stimulated monocytes by laser confocal microscopy. mbound CD14 (A to D) and intracellular CD14 (E to H) were detected by staining unstimulated monocytes (A, B, E, and F) and monocytes stimulated with LPS (1 μg/10⁶/ml) (C, G, D, and H) with antibodies as described in Materials and Methods. Cells were then treated with proteinase K (B, D, F, and H) to remove mCD14, fixed, and mounted on slides. Results are those from one representative experiment out of three performed with cells from different donors.

FIG. 3

Effects of kinase inhibitors on CD14 internalization viewed by laser confocal microscopy. Purified human monocytes (10⁶/ml) were first treated with SB203580 (5 μM) (A), U0126 (10 μM) (B), or a mixture of both inhibitors (C) and then stimulated with LPS (1 μg/10⁶/ml) as described in Materials and Methods. Intracellular CD14 was detected as described for Fig. 4. Results are those from one representative experiment out of three performed with cells from different donors.

FIG. 4

Effects of kinase inhibitors on intracellular IL-1α, IL-1β, and TNF-α production by SAC-stimulated monocytes. Purified human monocytes (10⁶/ml) were first treated with SB203580 (5 μM), U0126 (10 μM), or a mixture of both inhibitors and then cultured either in the absence (RPMI) or in the presence of SAC (100 μg/10⁶/ml). Intracellular cytokine production was assessed using specific anticytokine fluorescent MAbs. An isotypic control was used as negative control. Monocytes were gated using fluorescent anti-CD14 MAbs. At least 5,000 CD14⁺ events were acquired for intracellular cytokine analysis. Results are presented as representative dot plots. Reported numbers in each quadrant represented the percentage of cells that are positive compared to isotypic negative controls. Results are those from one representative experiment out of four performed with cells from different donors.

FIG. 5

Lack of CD14 internalization in SAC-stimulated monocytes viewed by laser confocal microscopy. mCD14 (A and B) and intracellular CD14 (C and D) were detected by staining unstimulated monocytes (A and C) and monocytes stimulated with SAC (100 μg/10⁶/ml) (B and D) with MAbs as described in Materials and Methods. Cells were then treated with proteinase K (C and D) to remove mCD14, fixed, and mounted on slides. For all confocal images presented, acquisition parameters were kept constant. Results are those from one representative experiment out of three performed with cells from different donors.