Human Dendritic Cells Express the Complement Receptor Immunoglobulin Which Regulates T Cell Responses

Abstract

The B7 family-related protein V-set and Ig containing 4 (VSIG4), also known as Z39Ig and Complement Immunoglobulin Receptor (CRIg), is the most recent of the complement receptors to be identified, with substantially distinct properties from the classical complement receptors. The receptor displays both phagocytosis-promoting and anti-inflammatory properties. The receptor has been reported to be exclusively expressed in macrophages. We now present evidence, that CRIg is also expressed in human monocyte-derived dendritic cells (MDDC), including on the cell surface, implicating its role in adaptive immunity. Three CRIg transcripts were detected and by Western blotting analysis both the known Long (L) and Short (S) forms were prominent but we also identified another form running between these two. Cytokines regulated the expression of CRIg on dendritic cells, leading to its up- or down regulation. Furthermore, the steroid dexamethasone markedly upregulated CRIg expression, and in co-culture experiments, the dexamethasone conditioned dendritic cells caused significant inhibition of the phytohemagglutinin-induced and alloantigen-induced T cell proliferation responses. In the alloantigen-induced response the production of IFNγ, TNF-α, IL-13, IL-4, and TGF-β1, were also significantly reduced in cultures with dexamethasone-treated DCs. Under these conditions dexamethasone conditioned DCs did not increase the percentage of regulatory T cells (Treg). Interestingly, this suppression could be overcome by the addition of an anti-CRIg monoclonal antibody to the cultures. Thus, CRIg expression may be a control point in dendritic cell function through which drugs and inflammatory mediators may exert their tolerogenic- or immunogenic-promoting effects on dendritic cells.

Keywords: T cells; complement receptor immunoglobulin (CRIg); cytokines; dendritic cells; dexamethasone; immunosuppression.

Figure 1

CRIg expression in MDDC and effects of dexamethasone. (A) CRIg isoform transcripts detected in DC. Agarose gel electrophoresis (2%) was used to visualize transcript variant amplicons generated from the cDNA of HMDC. Lanes are labeled 1, 2, 3, 4, and 5 representing each CRIg isoform with (–) to the right of each isoform lane indicating the respective no template controls. GAPDH was a positive control. Gels are representative of three experiments. (B) Gating strategy for CRIg expression on DC by flow cytometry. CD209⁺ cells were gated before assessing anti-CRIg antibody (6H8-PE) staining; representative histogram shown. (C) CRIg isoform expression by Western blot using anti-CRIg monoclonal antibody (clone 3C9). A representative blot of total protein stained with Ponceau S shows consistency of protein loading. Band intensity for each isoform (L, I, and S) over protein load was determined by densitometry. Data are expressed as fold-change over control DC (n = 3). (D) Relative CRIg mRNA expression as detected by qPCR, normalized to GAPDH, expressed as fold-change over control DCs (n = 3). (E) Relative CRIg cell-surface expression by flow cytometry. Data are expressed as fold-change in CRIg-PE (6H8) mean fluorescence intensity minus isotype control (IgG1) of treated over control DCs (n = 7); representative histogram shown. Data are presented as means ± SEM of experiments conducted with cells from different individuals. Significance levels are indicated by asterisks: *P < 0.05, ****P < 0.0001.

Figure 2

Effects of dexamethasone conditioned DCs and anti-CRIg monoclonal antibody on PHA- and allogeneic-induced T cell proliferation, cytokine production and generation of iTreg. (A) ³H-TdR incorporation (DPM) in autologous DC-T cell co-culture in the presence/absence of dexamethasone, anti-CRIg 6H8 or isotype control antibodies, and PHA. Data are presented as means ± SEM of 6 experiments conducted with cells from different individuals. (B) ³H-TdR incorporation (DPM) in allogeneic DC-T cell co-culture in the presence/absence of dexamethasone treated DCs, anti-CRIg 6H8 antibody or isotype control antibodies. Data are presented as mean ± SEM of 5 experiments. (C–H) Effects on cytokine production in allogeneic DC-T cell cultures. Data are presented as mean ± SEM of 4–5 experiments. (I) Effects on generation of iTreg in allogeneic DC-T cell cultures. Data are presented as mean ± SEM of 4 experiments. Note that normalized data for the above cytokine production and Treg generation is presented in Supplementary Figure 3. Significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

LT-α and IFN-γ decrease CRIg expression in DC. (A,D) DC were treated with varying concentrations of cytokines and the levels of CRIg mRNA determined after 24 h of culture. (B,E) In other experiments, CRIg isoform proteins were assessed by Western blot after cells were treated with 40 ng/ml of each of the cytokines. (C,F) The effects on cell surface expression of CRIg was examined after a similar treatment. Data are presented as means ± SEM of three experiments, each conducted with cells from different individuals. The blot image (E) was spliced to exclude intervening lanes that represent other treatments and the complete un-spliced blot can be found in Supplementary Figure 2. Significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

IL-4 and IL-13 down regulate CRIg expression in DC. (A,D) DC were treated with a dose range of the cytokines for 24 h and then examined for CRIg mRNA expression. (B,E) The levels of total CRIg isoform proteins measured by Western blot in DC treated with 20 ng/ml of cytokines. (C,F) Similarly treated DC were examined for surface expression of CRIg by flow cytometry. Data are presented as means ± SEM of at 3–4 experiments, each conducted with cells from a different individual. Significance levels are indicated by asterisks: **P < 0.01, ***P < 0.001, ****P < 0.0001, whilst n.s. indicates non-significance.

Figure 5

IL-10 and TGF-β1 increase CRIg expression in DC. (A,D) DC were treated with varying concentrations of the cytokines for 24 h and then examined for CRIg mRNA expression. DC were treated with either 40 ng/ml of IL-10 or 25 ng/ml of TGF-β1 and CRIg isoform protein expression in DC lysates (B,E) or CRIg expression on the cell surface (C,F) were measured. Data are presented as means ± SEM of three experiments, each conducted with cells from different individuals. The blot image in (E) was spliced to exclude intervening lanes that represent other treatments and the complete un-spliced blot can be found in Supplementary Figure 2. Significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, whilst n.s. indicates non-significance.

Figure 6

Effect of TNF-α, IL-1β, and IL-10 on CRIg expression in DC. (A,D,G) DC were treated with varying concentrations of the cytokines for 24 h and then examined for CRIg mRNA expression. For examination of CRIg isoform protein expression (B,E,H) and cell surface expression (C,F,I), the cells were treated with 40 ng/ml of cytokine. Data are presented as means ± SEM of three experiments, each conducted with cells from different individuals. Significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, whilst n.s. indicates non-significance.

Figure 7

M-CSF and GM-CSF increase CRIg in DC. (A,D) DC were treated with varying concentrations of the CSF and the CRIg mRNA expression determined. Changes in CRIg isoform proteins (B,E) and cell surface (C,F) CRIg expression on DC treated with 40 ng/ml of each CSF are shown. Data are presented as means ± SEM of three experiments, each conducted with cells from different individuals. Significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ****P < 0.0001, whilst n.s. indicates non-significance.