Article Figures & Data
Figures
- FIGURE 1.
Increased uptake of CRP-opsonized R36a by wild-type, but not by FcR γ-chain−/− DC. A, R36a were labeled with CM-DiI, treated with 100 μg/ml CRP for 30 min, and added to BMDC at a ratio of 800:1 (R36a:DC). Cells were incubated for 1 h, then washed and stained with FITC anti-CD11c. Uptake of R36a was determined by two-color flow cytometry. Results are presented as the CM-DiI fluorescence of the CD11c+ cells. Mean ± SEM of four experiments. B, The same experiment was done with the addition of 10−4 M PC where indicated. Mean ± SEM of two experiments. C, Representative two-color flow diagrams for C57BL/6 DC after incubation with R36a (left) or CRP-treated R36a (right).
- FIGURE 2.
Effect of CRP and R36a on DC maturation markers. R36a were incubated with 100 μg/ml CRP for 30 min and added to BMDC at a ratio of 800:1 (R36a:DC). DC were stained for maturation markers after 20 h. The geometric mean fluorescence intensity for each marker relative to the isotype control was determined. Results from three experiments were normalized to the unpulsed DC control to allow presentation of combined data. The markers were MHC class II (A), CD40 (B), CD80 (C), and CD86 (D). Significant differences between R36a and R36a + CRP are indicated by ∗, p < 0.05.
- FIGURE 3.
CRP opsonization increases IgG responses to PC and PspA in recipients of DC pulsed with R36a. BMDC from C57BL/6 mice were pulsed for 4.5 h with R36a or CRP-treated R36a and washed extensively to remove free bacteria. Recipient mice (n = 5/group) were injected with 106 DC i.p. on day 0. On day 28 (arrow), all mice were injected i.p. with 5 × 106 free R36a. Serum was collected weekly for Ab determinations by ELISA. Results are expressed as the absorbance readings multiplied by the serum dilution. Data were analyzed by one-way ANOVA. Significant differences between R36a and R36a + CRP groups are indicated by ∗∗, p < 0.01; ∗∗∗, p < 0.0001. Results are representative of two independent experiments using BMDC. A, IgM anti-PC response. B, IgG anti-PC response. C, IgG anti-PspA response.
- FIGURE 4.
CRP opsonization increases IgG responses to PC and PspA in recipients of Jaws II DC pulsed with R36a. The Jaws II DC line was pulsed for 4.5 h with R36a or CRP-treated R36a and washed extensively to remove free bacteria. C57BL/6 recipient mice (n = 5/group) were injected with 106 Jaws II DC i.p. on day 0. On day 28 (arrow), all mice were injected i.p. with 5 × 106 free R36a. Serum was collected weekly for Ab determinations by ELISA. Results are expressed as the absorbance readings multiplied by the serum dilution. Data were analyzed by one-way ANOVA. Significant differences between R36a and R36a + CRP groups are indicated by ∗∗, p < 0.01; ∗∗∗, p < 0.0001. Results are representative of two experiments using the Jaws II C57BL/6 DC cell line transferred into C57BL/6 recipients. A, IgM anti-PC response. B, IgG anti-PC response. C, IgG anti-PspA response.
- FIGURE 5.
CRP opsonization has no effect on Ab responses to PC and PspA in C57BL/6 recipients of FcR γ-chain−/− (N12) DC pulsed with R36a. BMDC from C57BL/6 FcR γ-chain−/− mice were pulsed for 4.5 h with R36a or CRP-treated R36a and washed extensively to remove free bacteria. C57BL/6 recipient mice (n = 5/group) were injected with 106 BMDC i.p. on day 0. On day 28 (arrow), all mice were injected i.p. with 5 × 106 free R36a. Serum was collected weekly for Ab determinations by ELISA. Results are expressed as the absorbance readings multiplied by the serum dilution. Data were analyzed by one-way ANOVA. No significant differences were seen between the R36a and R36a + CRP groups. Results are from one experiment using C57BL/6 FcR γ-chain−/− DC transferred into C57BL/6 recipients. Similar results were seen in two additional experiments using B6 × 129 FcR γ-chain−/− DC transferred into B6 × 129 recipients. A, IgM anti-PC response; B, IgG anti-PC response; and C, IgG anti-PspA response.
- FIGURE 6.
CRP opsonization increases late IgG responses to PC and PspA in C57BL/6 recipients of DC pulsed with R36a. Ab responses to PC and PspA in C57BL/6 recipients of C57BL/6 DC or B6 × 129 recipients of B6 × 129 FcR γ-chain−/− DC pulsed with R36a or CRP-R36a. DC were pulsed for 4.5 h with R36a or CRP-treated R36a and washed extensively to remove free bacteria. Recipient mice (n = 5/group) were injected with 106 DC i.p. on day 0. On day 28 (data not shown) and day 98 (arrow), all mice were injected i.p. with 5 × 106 free R36a. Serum was collected weekly for Ab determinations. Results are expressed as the absorbance readings multiplied by the serum dilution. Data were analyzed by one-way ANOVA. Significant differences were seen between the R36a and R36a + CRP groups in the recipients of wild-type (A and C), but not DC from FcR γ-chain−/− mice (B and D). ∗, p < 0.05; ∗∗, p < 0.01. A and B, IgG anti-PC response; C and D, IgG anti-PspA response.
- FIGURE 7.
CRP opsonization increases survival in recipients of DC pulsed with R36a challenged with Pn3. BMDC were pulsed for 4.5 h with R36a or CRP-treated R36a and washed extensively to remove free bacteria. Recipient mice were injected with 106 DC i.p. on day 0. On day 28, all mice were infected intranasally under anesthesia with 5 × 106 Pn3. A, Survival of C57BL/6 mice immunized with C57BL/6 DC that were unpulsed or pulsed with R36a or CRP-opsonized R36a. B, B6 × 129 mice immunized with BMDC from FcR γ-chain−/− mice that were unpulsed or pulsed with R36a or CRP-opsonized R36a. Two experiments were combined (n = 12/group). For the C57BL/6 mice, p < 0.05 for immunization with CRP-treated R36a vs either other group. For the recipients of FcR γ-chain−/− DC, there were no significant differences in survival among the three groups.
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