Cross-Linking Cell Surface Chemokine Receptors Leads to Isolation, Activation, and Differentiation of Monocytes into Potent Dendritic Cells

Abstract

Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic naïve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.

Gene Expression Profiling of Dendritic Cells Reveals Important Mechanisms Associated with Predisposition to Staphylococcus Infections

“…Among these down-regulated genes, the chemokine receptor ccr3, which is highly expressed in immature dendritic cells, and reported to be repressed in inflammatory conditions was found to be down-regulated [40]. By contrast, the transcription of markers of DC maturation ( cd40 and cd83 ) was increased after stimulation and clearly showed that maturation has occurred, whereas genes involved in cell to cell signaling and cell adhesion ( sulf2 and cdh1 ) were repressed.…”

Lipopolysaccharide induces the expression of an autocrine prolactin loop enhancing inflammatory response in monocytes

“…MCF-7 breast cancer cell line (ATCC®) was cultivated in RPMI 1640 as reported [24]. Monocytes from PBMC were isolated from heparinized (5 U/ml) blood of ten healthy male donors (29.8 ± 7.4 years old) by a standard density gradient centrifugation at 400 g using lymphocyte separation medium (Sigma Chemical) for 15 minutes at room temperature as described [25]. The cells at the interface were collected and washed three times in cold PBS containing 0.1% BSA.…”

Humanized Mice

“…Recently, the Balb/c Rag2 −/− g c −/− HIS mouse was used to study human disease, particularly infection by HIV Gorantla et al 2006;Zhang et al 2006), opening the road to developing new therapeutic approaches. However, HIS mice also provide an in vivo tool allowing for a more in-depth analysis of human lymphocyte development by providing access to human lymphocytes from tissues that are typically difficult to obtain.…”

“…In addition, we recently found a simple method to isolate monocytes from bulk PBMC by using antichemokine receptor monoclonal antibody-coated plates (Nimura et al 2006). A conventional method for generating DC from monocytes has been established, and there are commercially available kits for monocyte purification.…”