C1q deficiency leads to the defective suppression of IFN-alpha in response to nucleoprotein containing immune complexes - PubMed (original) (raw)
C1q deficiency leads to the defective suppression of IFN-alpha in response to nucleoprotein containing immune complexes
Deanna M Santer et al. J Immunol. 2010.
Abstract
Almost all humans with homozygous deficiency of C1q develop systemic lupus erythematosus (SLE). The precise cellular mechanism(s) by which C1q prevents the development of SLE remains unclear. In this study, we tested the role of C1q in the regulation of IFN-α induced by immune complexes (ICs) in vitro, as well as the consequences of lack of C1q in vivo. Our experiments revealed that C1q preferentially promotes the binding of SLE ICs to monocytes rather than plasmacytoid dendritic cells, but this inhibition was not due to the induction of inhibitory soluble factors. The presence of C1q also altered the trafficking of ICs within monocytes such that ICs persisted in early endosomes. In patients with C1q deficiency, serum and cerebrospinal fluid levels of IFN-α and IFN-γ-inducible protein-10 levels were elevated and strongly correlated with Ro autoantibodies, demonstrating the clinical significance of these observations. These studies therefore associate C1q deficiency with defective regulation of IFN-α and provide a better understanding of the cellular mechanisms by which C1q prevents the development of IC-stimulated autoimmunity.
Conflict of interest statement
Disclosures
The authors have no financial conflicts of interest.
Figures
FIGURE 1
C1q is required for serum inhibition of IFN-α induced by SLE ICs. A, SLE ICs were formed with diluted SLE serum (1:2000) and freeze-thawed Ag (1%) and added to IFN-primed PBMCs (IFN bioassay). Untreated or HI NHS was added at the time of IC addition at the concentrations indicated. B, Eight SLE sera were left untreated or HI and added with freeze-thawed Ag to IFN-primed PBMCs, as in A. Results are representative of two donors tested with all serum IC stimulations on the same day. Horizontal lines represent mean pg/ml IFN-α induced. The dashed line represents one SLE patient with low serum complement activity. C, The IFN bioassay was performed in the presence of NHS, C1q-depleted serum, or C2-depleted serum at the concentrations shown. D, The IFN bioassay was run in the presence of NHS or C1q dep serum reconstituted with purified C1q protein, as shown (hatched bars). IFN-α was quantified in supernatants by ELISA after 20 h and expressed as pg/ml or percentage of inhibition of IFN-α production relative to cells that were not treated with serum. Results are shown as the mean ± SEM of 3–13 (A), 3–12 (C), or 9–11 (D) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. C1q dep, C1q depleted; C2 dep, C2 depleted.
FIGURE 2
C1q itself inhibits IFN-α through interaction with SLE ICs at the time of stimulation. A, The IFN bioassay was performed as in Fig. 1, except that the PBMCs were not primed, and purified C1q (0.5–50 µg/ml) was added to the culture. B, C1q (50 µg/ml) was added to PBMCs that were either unprimed or IFN primed in the IFN bioassay. C, Isolated C1q (50 µg/ml) was added to unprimed PBMCs for 4 h at 37°C. All cells, even those not treated with C1q, were then extensively washed (+ Wash), SLE ICs were added, and the supernatants were collected after 20 h. In comparison, C1q was added simultaneously with ICs with no washing (No Wash). IFN-α was quantified and expressed as percentage of inhibition, as described in Fig. 1. Results are shown as the mean + SEM of four to seven independent experiments. Results in C are representative of four independent experiments. Data are shown with SLE ICs formed from two different patients’ serum in each figure (SLE ICs 1 and 2). **p < 0.01; ***p < 0.001.
FIGURE 3
C1q-deficient patient sera fail to inhibit IFN-α induction by SLE ICs and contain elevated levels of IFN-α and IP-10 that strongly correlate with Ro autoantibodies. A, Serum from normal or from siblings with (C1qD 1–4) or without (unaffected) C1qD was added (5% v/v) to the IFN bioassay in the presence or absence of purified C1q (5µg/ml). IFN-α was quantified by ELISA and expressed as percentage of inhibition, as in Fig. 1. Results are representative of three to four independent experiments. B, SLE ICs were formed with purified SLE IgG (75 or 150µg/ml) and freeze-thawed Ag (2% v/v), and then incubated with unaffected or C1qD sibling sera (patient 3 or 4, 5% v/v) and IFN-primed PBMCs. IFN-α was quantified by ELISA after 20 h. Results are representative of two independent experiments. C and D, IFN-α (C) and IP-10 (D) were quantified in sera or cerebrospinal fluid from normal, C1qD patients or their unaffected sibling, as described in Materials and Methods. The correlation between IFN-α and IP-10 levels in sera was statistically significant (Pearson correlation coefficient r = 0.9769, p = 0.004). E, Serum IFN-α levels and autoantibodies in patient sera or cerebrospinal fluid were measured by ELISA or autoantigen array, respectively, as described in Materials and Methods. A positive control of pooled autoreactive sera and a negative control of buffer alone were also included. Normalized MFIs for each autoantigen were calculated by dividing by the IgG concentration in each patient’s serum (mg/ml). Pearson correlation coefficients (r) are indicated with p values for only those correlations that were statistically significant. The diagonal lines show linear regression. bRo/SSA refers to purified bovine autoantigen, whereas Ro/SSA refers to recombinant human protein. N, normal; n.s., not significant; Unaff, unaffected.
FIGURE 4
CD14+ monocytes are required for C1q-dependent inhibition of IFN-α. A, SLE ICs with or without purified C1q (50 µg/ml) were incubated with isolated pDCs and IFN-α quantified after 20 h. The results are expressed as IFN-α concentration (mean ± SEM of 4 [SLE IC 1] or 9 [SLE IC 1] independent experiments). B, PBMCs were mock depleted (total PBMC) or depleted of CD14+ monocytes, CD19+ B cells, or CD56+ NK/NKT cells, as described in Materials and Methods, and stimulated by SLE ICs in the presence or absence of C1q (50 µg/ml). IFN-α concentrations were quantified as in Fig. 1 and are expressed as percentage of inhibition relative to IC-alone treatment and are representative of four independent experiments. C, The IFN bioassay was performed with total PBMCs, isolated pDCs or cocultures of pDCs, and isolated monocytes (pDC:monocyte ratios 1:20 or 1:40) in the presence or absence of C1q (50 µg/ml). Results of four independent experiments are expressed as percentage of inhibition relative to IC-alone treatment (mean + SEM), as in Fig. 1. CD14-dep, depleted of CD14+ monocytes; CD19-dep, depleted of CD19+ B cells; CD56-dep, depleted of CD56+ NK/NKT cells.
FIGURE 5
The presence of C1q favors SLE IC binding to monocytes. A, SLE ICs formed with Alexa Fluor 647 (AF647) –labeled SmRNP autoantigen and SLE serum (1:500) were incubated with PBMCs at 37°C in the presence or absence of C1q for 2 h and IC binding to pDCs and monocytes analyzed by flow cytometry. Representative FACS plots for IC binding to pDCs and monocytes are shown in the top panels, and percentage of IC+ and MFI for each cell type shown in the lower panels. Compiled results are shown as percentage of IC+ (compared with SmRNP-alone control) and MFI (MFI for pDCs is for within IC+ gate) and are expressed as the mean + SEM of five to nine independent experiments. B, SLE ICs with or without C1q (2.5 or 25 µg/ml) were incubated with isolated pDCs or with pDC-monocyte cocultures at a 1:30 pDC/monocyte ratio. The percentages of ICs associated with pDCs or monocytes within the pDC or pDC + monocyte cocultures are shown in circle or square symbols, respectively (% IC+). Dashed lines represent IC binding in coculture conditions. Representative results are shown from one of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. n.s., not significant.
FIGURE 6
C1q-containing ICs can bind to a CD32-independent receptor on monocytes and accumulate in early endosomes. A, Blocking Ab to FcγRII (CD32) or isotype control mouse IgG1 (Iso) was added to PBMCs for 45 min at 37°C before addition of AF647-labeled SLE ICs in the presence or absence of C1q. ICs were left to bind at 37°C for 2 h, and the percentage of IC+ pDCs or monocytes within PBMC cultures was quantified by flow cytometry. Results are expressed as mean + SEM of five to seven independent experiments. B, Labeled ICs, as in A, (red) were added to purified monocytes with or without C1q (50 µg/ml) for 1 or 4 h at 37°C. Abs to CD71 or lysosomal-associated membrane protein-1 (green) were added to stain early endosomes and lysosomes, respectively. Image collection and quantification of colocalization with SLE ICs were performed using an ImageStream flow cytometer. Results are expressed as the mean + SEM of three to four independent experiments. The three- to four-digit numbers on the brightfield images correspond to the specific cell image of >10,000 images acquired.
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