Mitogenicity of the recombinant mycobacterial 27-kilodalton lipoprotein is not connected to its antiprotective effect

Abstract

We reported previously that even though immunization with the recombinant mycobacterial 27-kDa lipoprotein (r27) induced a Th1-type response in mice, the vaccinated mice became more susceptible to challenge with Mycobacterium tuberculosis. In this study we show that r27 stimulates naive splenocytes to proliferate. Acylation of r27 was crucial for this effect, since a nonacylated mutant of r27, termed r27DeltaSP, failed to stimulate splenocytes either in vitro or in vivo. Depletion experiments indicated that only B cells were proliferating in a T-cell-independent manner. We also found that r27 is recognized by TLR2, which is involved in mitogenic stimulation. Interestingly, r27 but not r27DeltaSP induced high gamma interferon levels in splenocyte supernatants, whereas no significant interleukin-2 levels were detected. Since B-cell polyclonal activation might aggravate pathogen infection, we asked whether the antiprotective effect of the r27 lipoprotein is associated with its mitogenicity. We showed that, as in the case of r27, immunization of mice with the nonmitogenic r27DeltaSP lipoprotein resulted in increased M. tuberculosis multiplication. We conclude that the antiprotective effect of the r27 lipoprotein must be linked to properties of the polypeptide portion of the lipoprotein rather than to its lipid moiety and its mitogenicity.

FIG. 1.

Proliferative response of naive BALB/c splenocytes to r27. (A) BALB/c splenocytes (4 × 10⁵/well) were stimulated with various concentrations of r27. [³H]thymidine was added at different time points, and proliferation was measured after 16 h of incubation. (B and C) BALB/c splenocytes (B) or C3H/HeN and C3H/HeJ splenocytes (4 × 10⁵/well) (C) were stimulated with either r27 (10 μg/ml), LPS (5 μg/ml), or boiled r27 (10 μg/ml) for 24 h in the presence or absence of polymyxin B (5 μg/ml). After this period, [³H]thymidine was added for 16 h, and the proliferation response was measured. **, P < 0.001 (compared to stimulation with LPS and without polymyxin B); #, P < 0.001 (compared to stimulation with r27).

FIG. 2.

Acylation of r27 is crucial for its mitogenicity. (A) BALB/c splenocytes (4 × 10⁵/well) were stimulated with r27, r27ΔSP (10 μg/ml), or LPS (5 μg/ml) for 24 h in the presence or absence of polymyxin B (5 μg/ml). After this period, [³H]thymidine was added for 16 h, and the proliferation response was measured. #, P < 0.001 (compared to stimulation with r27); *, P < 0.01 (compared to the relevant non-polymyxin B-treated group). (B) Mice were injected intravenously with 25 μg of r27, r27ΔSP, or saline as a control. Five days after the injection, spleens were removed, and total splenocytes were counted by trypan blue exclusion. **, P < 0.001 (compared to r27-injected mice).

FIG. 3.

B cells proliferate in response to r27. (A) B or T cells were purified from BALB/c splenocytes and stimulated with r27 (10 μg/ml) for 24 h; then [³H]thymidine was added for an additional 16 h. **, P < 0.001 (compared to B cells). (B) IFN-γ levels were measured after 48 h of stimulation with r27 in whole BALB/c splenocytes, purified B or T cells, and whole nu/nu splenocytes. *, P < 0.01 (compared to stimulation with r27ΔSP).

FIG. 4.

r27 is recognized by TLR2. (A) RAW 264.7 cells (5 × 10⁵/well) were stimulated with various concentration of r27 or r27ΔSP in the presence of polymyxin B. After 24 h, culture supernatants were collected from the wells, and IL-12 levels were measured by ELISA. (B) Thioglycolate-elicited peritoneal macrophages from C3HEB/FeJ and C3H/HeJ mice were stimulated with LPS (10 ng/ml) or r27 (10 μg/ml), supernatants were collected after 24 h, and IL-12 secretion was measured by ELISA. (C) CHO/CD14 and CHO/CD14/TLR2 cells (5 × 10⁵/well) were stimulated with r27 or r27ΔSP (5 μg/ml) for 24 h. After this period, cells were collected, labeled with fluorescein isothiocyanate-conjugated anti-human CD25, and then analyzed by FACS. Similar results were obtained in four independent experiments.

FIG. 5.

Splenocyte proliferation induced by r27 is TLR2 dependent. BALB/c or C3H/HeJ splenocytes (4 × 10⁵/well) were incubated with TLR2 antisense (AS-ODN) or sense (S-ODN) oligonucleotides (5 μM) for 24 h. (A) After this period, mRNA was prepared from the cells, and TLR2 or β-actin mRNA levels were detected by RT-PCR and quantified by using TINA software. (B) Alternatively, after 24 h, r27 was added to the treated splenocyte cultures for another 24 h; then [³H]thymidine was added for 16 additional h, and the proliferation response was measured. *, P < 0.01 (compared to untreated splenocytes).

FIG. 6.

Immune response induced by the acylated form of the 27-kDa protein. Mice were immunized with either r27 or r27ΔSP, in the presence or absence of Ribi. (A) Antibody titers against r27 or r27ΔSP were measured for immunized mice by using the relevant immunizing antigen. OD₄₀₅, optical density at 405 nm. (B) IFN-γ secretion in splenocytes from immunized groups after stimulation with r27 or r27ΔSP. (C) DTH response to r27 or to r27ΔSP in M. tuberculosis-infected mice. **, P < 0.005 (compared to stimulation with r27).

FIG. 7.

Both r27 and r27ΔSP increase M. tuberculosis CFU numbers in immunized mice. BALB/c mice were immunized twice with 10 μg of r27 or r27ΔSP 2 weeks apart. After 4 weeks, mice were challenged with 5 × 10⁵ CFU of M. tuberculosis strain H37Rv; 5 weeks later, mice were sacrificed, and CFU in the spleen were determined. This experiment was repeated three times; results of one representative experiment are presented here. *, P < 0.05; **, P < 0.01 (compared to the naive group).