Newly recruited human monocytes have a preserved responsiveness towards bacterial peptides in terms of CD11b up-regulation and intracellular hydrogen peroxide production

Abstract

The transmigration of peripheral human monocytes to the interstitium is a fundamental step in the host-defence mechanism against infections. Little is known about the state of function of in vivo transmigrated interstitial monocytes prior to differentiation into macrophages and dendritic cells. We hypothesized that newly recruited interstitial monocytes have a preserved responsiveness against bacterial-related peptides, giving them a specific role in the immediate defence against invading pathogens. In order to test this hypothesis, we explored the responsiveness of in vivo transmigrated as well as peripheral monocytes, in terms of CD11b expression and H(2)O(2) production towards the bacterial-related peptide formylmethionylleucylphenylalanine (fMLP) by the use of a skin chamber technique. In addition, we analysed the concentration of interleukin (IL)-8, monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor (TNF)-alpha in the skin blister exudates and in the circulation. We demonstrate that in vivo-transmigrated monocytes had a fivefold higher CD11b expression compared to monocytes obtained from the peripheral circulation. fMLP exposure induced a significantly higher CD11b expression on transmigrated cells compared to peripheral monocytes. In addition, newly recruited monocytes had a preserved H(2)O(2) production. The interstitial concentration of IL-8, MCP-1 and TNF-alpha was significantly higher in blister exudates compared to that in the peripheral circulation. Thus, in vivo transmigrated human monocytes preserve their capacity to respond towards bacterial peptides in terms of CD11b up-regulation and H(2)O(2) generation. These data strengthen a role for newly recruited interstitial human monocytes in the immediate defence against invading pathogens.

Fig. 1

A representative two-parametric scatterplot and topographic histogram of peripheral circulation (a) and blister exudates (b) from a healthy subject, with the different leucocyte subpopulation. FS: forward scatter; SS: side scatter.

Fig. 2

Expression of CD11b on monocytes in the peripheral circulation and on monocytes in skin blister exudates, at 4°C (open bars) and following formylmethionylleucylphenylalanine activation (cross-hatching) in healthy subjects (n = 12). The CD11b expression was analysed by flow cytometry and expressed as mean fluorescence intensity. The results are presented as median and interquartile range. *P < 0·05.

Fig. 3

Hydrogen peroxide (H₂O₂) production, at rest (phosphate-buffered saline) and in response to formylmethionylleucylphenylalanine and phorbol-12-myristate 7-acetate stimulation in monocytes from the peripheral circulation and in skin chambers exudates in healthy donors (n = 11). The H₂O₂ production is analysed by flow cytometry and expressed as mean fluorescence intensity (MFI). The data are presented as median, interquartile range and outliers. *P < 0·05, **P < 0·01; ↑: two outliers (151 and 119 MFI, respectively).

Fig. 4

Concentration of (a) monocyte chemotactic protein-1 (ρg/ml) and (b) interleukin-6 (ρg/ml) in serum and at the intermediate and intense site of inflammation in healthy subjects (n = 11), analysed by enzyme-linked immunosorbent assay. Data are presented as median and interquartile range. *P < 0·05, **P < 0·01.

Fig. 5

Correlation between tumour necrosis factor-α concentration in the blister exudates and CD11b expression on transmigrated monocytes. Intermediate inflammation (n = 6) (r = 0·89, P < 0·05) and intense inflammation (n = 9) (r = 0·75, P < 0·05).