Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells

Abstract

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.

Figure 1

C is involved in the early phase of eliciting CS and DTH. (A) CBA/J mice (4/group) were actively contact sensitized with PCl applied topically to the shaved chest and abdomen. 4 d later the mice were skin challenged to elicit CS by painting both ears with PCl. The resulting CS ear swelling responses were measured with a dial caliper by comparing thickness before, vs. 2 and 24 h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2 and 24 h. (B) TNP-SRBC (10⁵ RBC) were injected i.v. to immunize BDF₁ mice (4/group) which were challenged s.c. 5 d later in both hind footpads with 10⁸ TNP-SRBC. Footpad swelling was measured with a dial caliper before and 24 h after challenge. In both A and B, sCR1 was injected (750 μg/mouse) i.p. 30 min before Ag challenge (groups B and D), or 3 h after challenge (group E). *P <0.05, **P <0.01, compared with PBS injected and immunized and Ag challenged controls (group C).

Figure 2

CS and DTH were inhibited by local depletion of C in ears by CVF injection. (A) CBA/J mice (4/group) were actively contact sensitized with PCl applied topically to the shaved chest and abdomen. 4 d later mice were skin challenged to elicit CS responses by painting both ears with PCl (groups C and D). Subsequent CS ear swelling responses were measured with a dial caliper by comparing thickness before vs. 2 and 24 h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2 and 24 h. PBS or CVF (5 μg/20 μl) was injected directly into the ears s.c. with a 30 gauge, 1/2′′ needle, 48 h before challenge (groups B and D). (B) TNP-SRBC (10⁵ cells) were injected i.v. to immunize BDF₁ mice (4/group) which were challenged s.c. 5 d later in both hind footpads with 10⁸ TNP-SRBC. Footpad swelling was measured with a dial caliper before and 24 h after challenge. PBS or CVF (1 μg/40 μl) was injected locally s.c. 48 h before footpad challenge. *P <0.05, **P <0.01, compared to 2- and 24-h positive immune and Ag-challenged controls which were injected with PBS (group C).

Figure 3

C5 depletion by anti-C5 mAb–inhibited CS. CBA/J mice (4/group) were actively contact sensitized with PCl applied topically to the shaved chest and abdomen. 4 d later, mice were skin challenged to elicit CS by painting both ears with PCl. CS ear swelling responses were measured with a dial caliper by comparing thickness before, vs. 2 and 24 h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2 and 24 h after challenge. (A) Systemic anti-C5 mAb (1 mg/mouse) was injected i.v. 24 and 4 h before ear challenge with Ag. (B) Local anti-C5 mAb (20 μg/20 μl) was injected s.c. into ears 24 h before ear challenge. *P <0.05, **P <0.01, compared with IgG₁ isotype control (group C).

Figure 4

Increased C5-titer in 24-h CS ear extracts. Contact sensitized mice were skin challenged topically on day 4 to elicit CS by painting both ears with PCl. 24 h later the challenged ears were extracted with PBS, as described in Materials and Methods. (A) Total protein concentration (mg/ ear) and (B) C5-titer per ear were determined. Closed and open circles represent two- and four-fold dilution of ear extracts with gelatin-veronal buffer, respectively. Each value per ear was expressed as a separate dot. The number of ear extracts in each group was 12, and was derived from 6 mice.

Figure 5

Anti-C treatment resulted in decreased chemotactic activity in 24-h CS ear extracts. Two different C inhibitors, anti-C5 mAb (A) and sCR1 (B), were used. Mice were ear challenged 4 d after PCl contact sensitization. Punch biopsies from each ear were collected 24 h later and then were extracted with PBS. In vitro chemotaxis assay was performed on the ear extracts using target J774A.1 macrophage cells which did not migrate against the RPMI 1640–0.25% gelatin medium. Human rC5a (100 ng/ml) and ZAMS (1:20) prepared from normal mouse serum were used as positive chemoattractant controls. (A) Mice were injected i.v. 24 and 4 h before ear challenge with either anti-C5 (1 mg/mouse) or with isotype (IgG₁) matched control anti–human C8 (1 mg/mouse). (B) Mice were injected i.p. with 125 μg/mouse sCR1 30 min before ear challenge.

Figure 6

Anti-C5 mAb treatment also decreased IFN-γ in 24-h CS ear extracts. Mice (4/group) were actively contact sensitized with PCl applied topically to the shaved chest and abdomen. 4 d later mice were skin challenged to elicit CS by painting both ears with PCl. Punch biopsies from each ear were collected 24 h later and were extracted with PBS. IFN-γ production in the ear extracts was determined by quantitative sandwich ELISA. (A) Systemic anti-C5 mAb (1 mg/mouse) was injected i.v. 24 and 4 h before ear challenge. (B) Local anti-C5 mAb (20 μg/20 μl) was injected s.c. into the ear 24 h before challenge. The number of mice in each group was four and eight samples of ear extracts in each group were pooled (A) or were assayed separately (B). *P <0.001 compared with isotype (IgG₁) controls.

Figure 7

Cell infiltration in 24-h CS ears was decreased by systemic treatment with anti-C5 mAb. Mice were immunized by painting with vehicle (a and b) or 5% PCl (c–f   ) on day 0, and challenged on the ears with PCl on day 4. Then, saline (a, c, and e) or 1 mg anti-C5 mAb (b, d, and f   ) was injected i.v. at 4 and 24 h before ear challenge. 5-μm sections of ears were obtained 24 h after Ag challenge, and were stained with hematoxylin and eosin. Magnification is 200 (a–d) or 1,000 (e and f  ). Leukocyte infiltration in CS era responses (c) was decreased by anti-C5 mAb treatment (d). Intraepidermal abscesses of leukocytes were observed frequently in ears from immune and challenged mice treated with saline (c and e), but not in ears of mice treated with anti-C5 mAb (d and f   ). Arrowheads in (c) denote intraepidermal abscess.

Figure 8

Quantitation of decreased cell infiltration in CS ears of mice treated with anti-C5 mAb. Sections of ears, as in Fig. 4, were histologically evaluated blindly. (A) Infiltration of leukocytes was graded from one to four, where one through four are, respectively, no infiltration, slight infiltration, modest infiltration, and significant infiltration. (B) Formation of intraepidermal abscesses was graded from one to three where they are, respectively, none seen, few seen, and many seen. Numbers in parentheses denote mean ± SE of histologic scores for each portion of the figure. *P <0.02 and **P <0.002, compared to positive controls (5% PCl-immunized, PBS-injected, ear-challenged group).

Figure 9

Impaired CS responses in B cell–deficient μMT mice. CS was induced in relatively hyporeactive C57Bl/6 control mice (groups A and B), and in C57Bl/6-Igh-6 (μMT) B cell–deficient mice (groups C and D) by topical application of PCl on days 0 and 1. Then, 6 d later the mice were skin challenged to elicit CS by painting both ears with 0.8% PCl. The resulting CS ear swelling responses were measured with a dial caliper by comparing thickness before, versus 2 and 24 h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2 and 24 h after challenge. Number of mice in each group was eight. *P <0.01, compared with C57Bl/6 mice controls (group B).