Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes - PubMed (original) (raw)

Comparative Study

Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes

T Spoettl et al. Clin Exp Immunol. 2006 Jul.

Abstract

Monocytes (MO) migrating into normal, non-inflamed intestinal mucosa undergo a specific differentiation resulting in a non-reactive, tolerogenic intestinal macrophage (IMAC). Recently we demonstrated the differentiation of MO into an intestinal-like macrophage (MAC) phenotype in vitro in a three-dimensional cell culture model (multi-cellular spheroid or MCS model). In the mucosa of patients with inflammatory bowel disease (IBD) in addition to normal IMAC, a reactive MAC population as well as increased levels of monocyte chemoattractant protein 1 (MCP-1) is found. The aim of this study was to investigate the influence of MCP-1 on the differentiation of MO into IMAC. MCS were generated from adenovirally transfected HT-29 cells overexpressing MCP-1, macrophage inflammatory protein 3 alpha (MIP-3alpha) or non-transfected controls and co-cultured with freshly elutriated blood MO. After 7 days of co-culture MCS were harvested, and expression of the surface antigens CD33 and CD14 as well as the intracellular MAC marker CD68 was determined by flow-cytometry or immunohistochemistry. MCP-1 and MIP-3alpha expression by HT-29 cells in the MCS was increased by transfection at the time of MCS formation. In contrast to MIP-3alpha, MCP-1 overexpression induced a massive migration of MO into the three-dimensional aggregates. Differentiation of IMAC was disturbed in MCP-1-transfected MCS compared to experiments with non-transfected control aggregates, or the MIP-3alpha-transfected MCS, as indicated by high CD14 expression of MO/IMAC cultured inside the MCP-1-transfected MCS, as shown by immunohistochemistry and FACS analysis. Neutralization of MCP-1 was followed by an almost complete absence of monocyte migration into the MCS. MCP-1 induced migration of MO into three-dimensional spheroids generated from HT-29 cells and inhibited intestinal-like differentiation of blood MO into IMAC. It may be speculated that MCP-1 could play a role in the disturbed IMAC differentiation in IBD mucosa.

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Figures

Fig. 1

Fig. 1

Chemokine levels in supernatants of transfected cells. (a) Monocyte chemoattractant protein (MCP)-1 levels in the supernatants of non-transfected controls after 48 h of culture were 40 ± 34 pg/ml compared to 1932 ± 1631 pg/ml in HT-29 cells 48 h after Ad5_MCP-1 transfection. In supernatants of spheroids cocultured with monocyte/macrophage (MO/MAC) for 7 days the mean value of MCP-1 in the supernatant was 1016 ± 970 pg/ml in non-transfected controls compared to 4645 ± 100 pg/ml in experiments with Ad5_MCP-1-transfected cells. ▪, Control; □, Ad5_MCP-1 transfected. (b) The mean value of macrophage inflammatory protein 3 alpha (MIP-3α) in the supernatants of HT-29 cells before generation of spheroids was 8·7 ± 7·9 pg/ml in non-transfected control cells compared to 58·1 ± 23·9 pg/ml in MIP-3α-transfected cells. MIP-3α values in supernatants of multi-cellular spheroid (MCS) after 7 days of co-culture with MO/MAC were 243 ± 214 pg/ml in aggregates consisting of non-transfected HT-29 cells and 448 ± 353 pg/ml in aggregates consisting of Ad5_MIP-3α-transfected cells. ▪, Control; □, Ad5_MCP-3α transfected.

Fig. 2

Fig. 2

Macrophages (MAC) inside the aggregates were identified by positive staining for the monocyte (MO)/MAC specific surface marker CD33. (a) CD33+ MO/MAC cultured in non-transfected, Ad5_Null- and macrophage inflammatory protein 3 alpha (MIP-3α)-transfected HT-29 multi-cellular spheroid (MCS) for 7 days. Only a low number of CD33+ cells can be detected. (b) CD33+ MO/MAC in Ad5_MCP-1-, Ad5_Null/Ad5_MCP-1- and Ad5_MIP-3α/Ad5_MCP-1-transfected HT-29 MCS after 7 days of culture. Clearly, many more CD33+ cells can be isolated from the MCS if monocyte chemoattractant protein (MCP)-1 was overexpressed.

Fig. 3

Fig. 3

Percentage of CD33+ monocyte/macrophage (MO/MAC) in multi-cellular spheroid (MCS) after 7 days of co-culture. In control experiments with spheroids from non-transfected HT-29 cells 4·5% of total cells could be identified as MO/MAC (CD33+ cells) after 7 days (n = 11) compared to 30·9% MO/MAC of total cells in aggregates from Ad5_MCP-1-transfected cells (n = 10). In spheroids generated from HT-29 cells transfected with the empty control virus 5·1% of total cells were MO/MAC (n = 4), co-transfection of HT-29 cells with Ad5_Null and Ad5_MCP-1 resulted in 37·9% of MO/MAC inside the aggregates (n = 4). Experiments with MCS from HT-29 cells transfected with Ad5_MIP-3α revealed 4·9% CD33+ cells inside the aggregates after 7 days of co-culture (n = 7), co-transfection with Ad5_macrophage inflammatory protein 3 alpha (MIP-3α) and Ad5_MCP-1 resulted in 36·1% of CD33+ cells inside the aggregates. □, Alone; ▪, with monocyte chemoattractant protein (MCP)-1.

Fig. 4

Fig. 4

Immunohistochemical staining of multi-cellular spheroid (MCS) co-cultured with monocytes (MO) for 7 days. Aggregates were stained for the intracellular MO/macrophage (MAC) specific marker CD68 and the differentiation and activation associated surface antigen CD14. (a–d) Non-transfected control MCS; (e–h) Ad-MCP-1-transfected MCS. (a) In non-transfected control MCS CD68+ MAC could be identified inside the aggregates after 7 days of co-culture; (b) no expression of CD14 could be detected in theses cells; (c) staining of EP4 revealed the epithelial character of the vast majority of cells; (d) isotype control staining; (e) transfection of HT-29 cells with Ad_MCP-1 resulted in a higher rate of monocyte migration into the aggregates; (f) a much higher number compared to non-transfected MCS expressed CD14; (g) EP4 staining; (h) isotype control (original magnification ×400).

Fig. 5

Fig. 5

Dot-plots of CD33/CD14 double-positive cells. (a) In non-transfected control spheroids 1·3% of total cells showed expression of CD33 and CD14. Transfection of HT-29 cells with Ad5_Null also resulted in 1·3% CD33+/CD14+) cells. In spheroids consisting of HT-29 cells transfected with Ad5_macrophage inflammatory protein 3 alpha (MIP-3α) 1·2% of total cells were positive for CD33 and CD14. A small population of CD33+/CD14– intestinal-like macrophages was present in all analyses (lower right quadrant, arrows). (b) In spheroids from Ad5_MCP-1-transfected cells 26·2% of total cells showed expression of CD33 and CD14 after 7 days. Co-transfection with Ad5_Null and Ad5_MCP-1 resulted in similar effects with 30·8% of total cells co-expressing CD33 and CD14. Co-transfection with Ad5_MIP-3α and Ad5_MCP-1 resulted in 34·6% of total cells expressing CD33 and CD14. No CD33+/CD14– population of cells can be detected.

Fig. 6

Fig. 6

Percentage of CD14+ cells with respect to CD33+ cells after 7 days of co-culture. The amount of CD14+ cells among CD33+ cells was significantly higher in Ad5_MCP-1-transfected spheroids compared to aggregates from non-transfected control cells (P = 0·002, _t_-test). The same result was obtained with monocytes (MO) cultured in Ad5_Null (40·0%) and AD 5_Null/Ad5_MCP-1 (85·5%) transfected aggregates (P = 0·001, _t_-test). Also comparison between MO cultured inside Ad5_macrophage inflammatory protein 3 alpha (MIP-3α) and Ad5_MIP-3α/MCP-1 aggregates revealed a significantly higher percentage of CD14+ MO/macrophage (MAC) among CD33+ MO/MAC when HT-29 cells were transfected with Ad5_MCP1 (43·4%versus 82·6%, P = 0·013, _t_-test). □, Alone; ▪, with monocyte chemoattractant protein (MCP)-1.

Fig. 7

Fig. 7

Neutralization of monocyte chemoattractant protein (MCP)-1 by the addition of anti-MCP-1 antibodies to the multi-cellular spheroid (MCS) model. A MCP-1 neutralizing antibody (R&D systems, cat. number AB-279-NA) was added to the cultures at days 1, 3 and 5 of the culture period. MCS were analysed by FACS analysis (a, b) and by immunohistochemistry (c, d). Whereas there was a high number of CD14 positive cells after transfection of HT-29 cells with Ad5_MCP-1 (a) neutralization of MCP-1 almost completely abrogated the presence of CD14+ cells in the MCS (b). For immunohistochemistry CD14 (Pharmingen, Heidelberg, Germany) was stained with APAAP (red) and CD68 (Dako; clone: KP1) was stained with BDHC (blue). The presence of CD68/CD14+ cells after Ad5_MCP-1 transfection was obvious (c), whereas no CD68/CD14 double-positive and almost no CD68 or CD14 single-positive cells could be observed after neutralization of MCP-1 (d). The experiment shown was conducted in triplicate and is representative of two further experiments.

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