TLR4-mediated signaling induces MMP9-dependent cleavage of B cell surface CD23

Abstract

IgE production is inversely regulated by circulating and B cell surface levels of the low affinity IgE receptor, CD23. To begin to understand physiologic determinants of CD23 expression, we analyzed effects of BCR and TLR stimulation on CD23 levels. BCR and TLR 2, 3, 4, 6, and 9 agonists induced CD23 down-modulation from the cell surface. However, among the ligands only TLR4 agonists induced transcriptional activation of CD23 and generation of significant soluble CD23. These responses were induced by LPS both in vitro and in vivo, and were seen in both murine and human B cells. LPS also induced expression of matrix metalloprotease 9 (MMP9) and failed to induce CD23 cleaving activity in MMP9(-/-) cells, thus implicating MMP9 in the LPS-induced release of CD23 from the cell surface. Finally, type 1 transitional B cells uniquely produce MMP9 in response to LPS, suggesting a mechanism wherein endotoxin induces T1 cell expression of MMP9, which mediates cleavage of CD23 on distinct, mature B cells.

FIGURE 1

Agonists of TLRs 2, 3, 4, 6, and 9 and the Ag receptor induce CD23 down modulation from B cells, but only LPS treatment results in detectable accumulation of soluble CD23 in culture supernatants. a, Surface expression CD23 by splenic B cells from C57BL/6 and MyD88-deficient mice was determined by flow cytometry 24 h after treatment with the indicated stimuli; solid line-untreated C57BL/6, shaded line-treated C57BL/6, dashed line-untreated MyD88^−/−, dotted line-treated MyD88^−/−. b, Release of CD23 and CD21 from the cell surface was measured by ELISA of culture supernatants from C57BL/6 () and MyD88^−/− (□) splenic B cells 24 h after treatment with the indicated stimuli. Statistical significance was determined using the Wilcoxon signed-rank test. The p value was adjusted for multiple comparisons using the false discovery rate approach.

FIGURE 2

LPS induces CD23 down-modulation by human B cells and the accumulation of sCD23 in vivo. C57BL/6 and MyD88-deficient mice were injected i.p. with 50 μg of LPS. Surface expression of CD23 on splenic B cells (a) and the average percent increase in sCD23 accumulation in the blood of mice immunized with LPS compared with PBS controls; solid line-untreated C57BL/6, shaded line-treated C57BL/6, dashed line-untreated MyD88^−/−, dotted line-treated MyD88^−/− (b) was determined 30-h post immunization; C57BL/6 () and MyD88^−/− (□). c, Human peripheral blood from three patients (◆, patient 1; ▲, patient 2; ■, patient 3) was incubated in the presence of the indicated concentrations of LPS for 24 h. CD23 surface expression on B cells (CD19⁺) was determined by flow cytometry. d, Supernatants from the cells cultured with 10 ng/ml LPS in c were analyzed by ELISA as described in the Materials and Methods. Shown is the average percent increase in sCD23 in LPS treated cultures relative to unstimulated controls. Statistical significance was determined using the Wilcoxon signed-rank test. *, p < 0.05.

FIGURE 3

LPS induces transcriptional activation of CD23 expression. a, C57BL/6 splenic B cell surface expression of CD23 was determined by flow cytometry following stimulation in vitro for the times indicated; untreated (●), LPS (■), or anti- μ (▲). b, New surface CD23 was determined by flow cytometry 6 h after in vitro treatment in the presence and absence of 12.5 μM actinomycin-D (left) or 100 ng/ml cyclohexamide (right). c, Relative cellular CD23 mRNA expression was quantified by real time PCR 6 and 24 h following treatment of the indicated stimuli. Statistical significance was determined using the Wilcoxon signed-rank test. (*, statistically significant change in CD23 expression compared with nontreated or treated cells without the inhibitor; **, statistically significant change in CD23 expression compared with the untreated control. p < 0.05).

FIGURE 4

LPS induced modulation of CD23 from the B cell surface is dependent on MMP9 expression. a, Splenocytes from C57BL/6 mice were preincubated for 1 h in presence or absence of 12.5 μM actinomycin-D (left) or 10 μM GM6001 (right) then stimulated as indicated. Soluble CD23 in supernatants was measured by ELISA 24 h after treatment. Relative MMP9 (b) and ADAM10 (c) expression was quantified by real time PCR of treated B220⁺ B cells and B220⁻ B cells. Statistical significance was determined using the Wilcoxon signed-rank test. p < 0.05. d, Release of CD23 cleaving factors was assayed by transfer of culture supernatants from wild-type (left histogram) or MMP9-deficient (right histogram) untreated (solid black line) or LPS treated (shaded) splenocytes to MyD88-deficient B cell cultures, followed by measurement of cell surface CD23 on these cells by flow cytometry.

FIGURE 5

Transitional 1 and non-B cells produce MMP9 in response to LPS treatment. a, CD23 surface expression was determined by flow cytometry 24 h after LPS treatment of the indicated B cell populations. b, Culture supernatants from LPS treated (filled line) or nontreated (solid line) C57BL/6 isolated splenic populations (shown on top of the histograms) were assayed for CD23 cleaving activity as in Fig. 4. Shown are histograms of supernatant treated MyD88^−/− B cells. c, LPS-treated culture supernatants from the indicated isolated populations were assayed as in Fig. 4 and data presented as mean CD23 expression by supernatant treated MyD88^−/− B cells.

FIGURE 6

IL-4 stimulation does not affect CD23 modulation in response to TLR4 and BCR ligands. Splenocytes from C57BL/6 mice were cultured for 24 h with or without IL-4 (20 ng/ml) plus F(ab′)₂ anti-IgM (1 μg/ml) or LPS(1 μg/ml). CD23 expression by B220⁺ cells was assayed by flow cytometry.