Evidence that a C1q/C1qR system regulates monocyte-derived dendritic cell differentiation at the interface of innate and acquired immunity

Abstract

Growing evidence shows that C1q modulates the growth and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because C1q regulates both innate and acquired immune responses, we postulated that C1q modulates the transition from monocytes to DCs, i.e. the interface between innate and acquired immunity. Human peripheral blood monocytes cultured with soluble C1q and DC growth factors (granulocyte-macrophage colony-stimulating factor + Interleukin-4) failed to down-regulate monocyte-associated (CD14, CD16) and up-regulate DC-associated (CD83, CD86) markers. Impaired DC differentiation was not due to apoptosis; further analysis revealed the development of CD14(hi)CD11c(hi)CD16 (+/-) cells that have previously been associated with both innate and acquired immunity. Monocyte-DC precursors expressed gC1qR, the receptor for globular heads of C1q, from the outset, while cC1qR, the receptor for the collagen tails of C1q, was expressed at low levels. Notably, the binding pattern of monoclonal antibodies specific to the globular heads of C1q indicated that C1q is bound to monocytes via globular heads, presumably through gC1qR. Moreover, gC1qR levels decreased, while cC1qR levels were dramatically amplified as monocytes differentiated into immature DC. Thus, specific C1q/C1q receptor (R) interactions may control the transition from the monocyte state (innate immunity) toward the professional antigen-presenting cell state (adaptive immunity).

Fig. 1

Kinetics of extracellular C1q expression during the monocyte-to-DC transition (surface [A,C], secreted [B]). Mononuclear cells (MNCs) isolated from PB and cultured in the presence of GM-CSF + IL-4 were analyzed for surface expression and secretion of C1q. (A) Cells were analyzed by flow cytometry for surface bound C1q on days 0–4. Cell surface expression of C1q was highest on days 0–2, and it was greatly reduced by day 4. Isotype-matched Ab was used as a negative control; cells were gated on the HLA-DR⁺ population. (n = 4) (B) Cell supernatants were assessed for secreted C1q by sandwich ELISA. C1q secretion remained at a steady level of 90 ± 3 ng/ml throughout days 1–4. (n = 4). (C) Detection of surface C1q on fresh PB monocytes by immunofluorescent microscopy. MNCs were isolated and immediately analyzed for surface bound C1q. Isotype-matched Ab was used as a negative control (n = 3). (D) Light scatter profile analysis of peripheral blood mononuclear cells on days 1–4 in culture in the presence of DC growth factors. One representative experiment is shown. The gated populations indicated (HLA-DR⁺) were used for subsequent analysis (n = 20). FSC, forward scatter; SSC, side scatter.

Fig. 2

C1q sustains CD14 expression on monocyte-derived DCs in culture. MNCs isolated from PB by density gradient centrifugation were cultured in the presence of GM-CSF + IL-4 ± 25 μg/ml C1q (A,B), or without the addition of cytokines ± 25 μg/ml C1q (D). For the dose-response analysis, several concentrations of C1q were added as indicated (C). Cells were analyzed on days 0–4 for the expression of CD14. (A) Dendritic cells cultured in the presence of C1q retained CD14 on their surface and maintained elevated CD14 expression until day 4. The numbers in the upper corners of the plots represent percentage of positive cells/MFI. One representative experiment illustrated by dot plot analysis is shown. (B) Temporal analysis performed by flow cytometry revealed significantly higher levels of CD14 expression in C1q treated cultures (G4 + C1q) compared to G4 alone until day 4. *P < 0.05, **P < 0.01 (n = 4) (C) Dose-response analysis confirmed that the MFI of CD14⁺ cells correlates positively with increasing doses of C1q on day 2 (n = 3). (D) Baseline CD14 expression (day 0) on monocytes increased after 24 h of C1q treatment without the addition of DC growth factors (n = 3). Cells were gated on the HLA-DR⁺ population for all experiments.

Fig. 3

C1q induced changes in DC differentiation are not due to selective DC death. Mononuclear cells were isolated from PB and cultured with or without 25 μg/ml C1q. Cells were collected on days 1–4 and analyzed for the co-expression of CD14 and annexin V. (A) Annexin V analysis revealed that monocyte-DCs cultured in the presence of C1q did not have higher percentage apoptosis of CD14⁻ or CD14⁺ cells than those cultured in G4 alone on day 3. A typical experiment is shown illustrated by dot plots gated on DR⁺ cells (n = 3). (B) Microscopic observation of monocyte-DCs cultured in the presence of C1q (right panel) did not show increased number of apoptotic cells by Wright stain analysis. C1q-treated cells (right panel) were smaller in size and displayed monocyte-like morphology, lacking the typical extending membrane processes (arrow) characteristic of iDCs developing in the presence of GM-CSF + IL-4 alone (left panel).

Fig. 4

C1q alters GM-CSF + IL-4 induced DC differentiation. Monocyte-DCs were isolated and cultured in the presence of GM-CSF + IL-4 (G4) and with or without 25 μg/ml C1q (A–C). For the dose-response experiments, several concentrations of C1q were added as indicated (D). (A) C1q significantly decreased CD86 expression in monocyte-DCs compared to G4. *P <0.05, **P <0.01 (n = 4). (B) Monocyte-DCs cultured in the presence of C1q showed a decrease in the percentage of CD83⁺ cells in comparison with cells cultured in G4 alone. While CD83 expression was detected on the surface of these cells, their MFI remained low throughout the days with or without the addition of C1q (data not shown). *P <0.05 (n = 4). (C) CD11c expression was increased by day 2 with the addition of C1q compared to day 0. There was no significant difference in CD11c expression levels on cells cultured with or without C1q (n = 6). (D) Dose-response analysis revealed that there was little or no difference in the distribution of HLA-DR between C1q treated versus untreated cells on day 2 (n = 3). Cells were gated on HLA-DR⁺ cells for all experiments.

Fig. 5

C1q promotes the development of distinct iDC subsets and increases phagocytic capacity of iDCs. Immature DCs were generated from PB cultured in the presence of GM-CSF + IL-4 ± 25 μg/ml C1q. (A) Multiparametric analysis reveals increased expansion of CD14^hiCD16^+/− cells on days 1 and 2 in the presence of C1q. These cells represent distinct subsets of iDCs. While CD14 expression was sustained until day 4, CD16 levels decreased by day 3 (data not shown). The numbers in the upper left and lower right quadrants of the plots represent percentage positive/MFI values of CD14 and CD16, respectively; the numbers in the upper right corners indicate percentage positive cells co-expressing CD14 and CD16 (n = 4). (B) There was little or no difference in MFI values with or without C1q on days 1 and 2, and 100% of the cells were CD11c^hi with C1q treatment. The numbers in the dot plots represent MFI values of CD11c. One typical experiment is shown; cells were gated on HLA-DR⁺ cells (n = 4). (C) C1q treatment significantly increased phagocytic uptake of fluorescein-labeled dextran on day 3. Monocytes were cultured for 3 days in the presence of GM-CSF + IL-4 (G4) ± 25 μg/ml C1q. On day 3, C1q-treated cells showed significantly increased endocytic capacity than cells cultured with DC growth factors alone (n = 7). *P <0.05 represents significant difference as compared to control.

Fig. 6

Varied expression of C1q receptors and specific binding orientation of surface bound C1q on monocyte-DC precursors may regulate DC differentiation events. Mononuclear cells cultured in the presence of GM-CSF + IL-4 were analyzed for the expression of cC1qR (A,C) and gC1qR (B,D) expression, and C1q binding orientation (E). (A) The percentage of cC1qR expression was variable on monocytes, but by day 2 nearly all monocyte-DCs had the receptor on their surface (n = 4). (B) On day 0, gC1qR was present on almost all the cells, and its expression was only slightly reduced by day 4 (n = 4). (C) Mean fluorescence analysis revealed that cC1qR expression was dramatically amplified by days 3 and 4 (n = 4). (D) Mean fluorescence intensity of gC1qR remained at relatively steady levels throughout the days (n = 4). (E) C1q is bound to the monocyte and DC surface via its globular head regions, while on M-CSF treated monocyte-macrophages its orientation is reversed. Binding orientation of C1q was determined using monoclonal antibodies specific to the globular head regions of C1q as well as polyclonal antibodies to the whole protein, and assessed by flow cytometry (n = 3). Experiments were gated on HLA-DR⁺ cells. *P<0.05, **P<0.01.