Exposure to mycobacteria primes the immune system for evolutionarily diverse heat shock proteins

Abstract

During stress conditions, such as infection, the synthesis of heat shock proteins (HSPs) in microorganisms is upregulated. Since a high degree of homology exists within each HSP family, we postulated that exposure to microorganisms could prime the immune system for evolutionarily diverse HSPs. We tested this hypothesis by priming mice with three microorganisms, namely, Mycobacterium bovis BCG, Mycobacterium vaccae, and Chlamydia pneumoniae. After this, mice received a dose of the various HSPs. We found that BCG and M. vaccae but not C. pneumoniae primed the immune system for the induction of secondary immunoglobulin G (IgG) responses to most of the HSPs tested. Analysis of the IgG1 and IgG2a profile and gamma interferon production induced against the HSPs revealed the induction of a mixture of responses. We also observed that sera from mice treated with M. vaccae and HSP70 were cross-reactive, but no antibody complexes were observed in their kidneys, which frequently are targets for autoantibody reactions. Our findings add further support for the use of HSPs as effective vaccine adjuvants.

FIG. 1.

Induction of IgG antibodies specific to Pf70C (a) and EB200 (b) in BALB/c mice primed with live (L) or heat-killed (HK) BCG or with the recombinant protein Pf70C-EB200. On day 0, mice were primed i.p. either with Pf70C-EB200 (▴) or with 10⁶ CFU live (⋄) or heat-killed (░⃞) BCG followed by boosting with 25 μg Pf70C-EB200 given i.p. 3 weeks later. Mice in the control group were immunized with only Pf70C-EB200 (○) once. Bleeding was done 7 days after boosting with the protein. The production of IgG antibodies specific for Pf70C and EB200 was evaluated by ELISA using 2.0 μg/ml of recombinant Pf70C and a cocktail of six synthetic peptides (1 μg/ml of each peptide) to coat the plates. Data are the mean optical density (OD) values (A₄₀₅ nm) and SD of individual sera of three mice per group, serially diluted in the wells. We show one representative result of three independent experiments. Background reactivity of the sera with BSA at the corresponding dilution was subtracted.

FIG. 2.

(a) Analysis of Pf70C-specific IgG1 and IgG2a isotype levels in sera of mice primed with live (L) BCG. Serial dilutions of sera of individual mice were studied. The results from the 1:400 dilutions are presented, showing the mean OD values (A₄₀₅ nm) and SD of results from three mice. Statistical analysis showed significant differences between the groups at P < 0.005 compared with the respective control group (Student's t test). Background reactivity of the sera with BSA at the corresponding dilution was subtracted. (b) In vitro production of IFN-γ by splenocytes derived from mice in response to Pf70C-EB200 or PPD. Spleens from live BCG-primed (not boosted) mice or nonimmunized mice were collected 5 weeks after priming. Splenocytes from individual mice were harvested. Single-cell preparations were stimulated separately with 10 μg/ml of Pf70C-EB200 or PPD, cultured (5 × 10⁵ cells/well) in triplicate in 96-well cell culture plates, and incubated for 72 h at 37°C in 5% CO₂. Cell supernatants were collected, pooled, and assayed for the presence of IFN-γ by ELISA. Values are the means and SD of results derived from three different spleens.

FIG. 3.

(a) HSP-specific IgG responses induced in BALB/c mice primed with live (L) BCG and boosted with various HSPs once. Control mice received only HSPs once. Serum samples were obtained from the mice 7 days after the protein immunization. The presence of HSP-specific antibodies was detected by ELISA using serial dilutions of individual sera from three mice. Significant differences between groups following a Student's t test are depicted with asterisks (*, P < 0.005 with the respective control group). (b) HSP-specific IgG1/IgG2a profile. Titers are expressed as the log₁₀ of the reciprocal of the highest dilution at an OD of 1.0. Background reactivity of the sera with BSA at the corresponding dilution was subtracted. (c) In vitro IFN-γ production by the splenocytes stimulated with various HSPs from mice primed with live BCG or not primed. Splenocytes of individual mice from each group were stimulated separately in triplicate with 10 μg/ml of each HSP and with 2 μg/ml of ConA (positive control) in 96-well cell culture plates and incubated for 72 h at 37°C in 5% CO₂. Cell supernatants were collected, pooled, and assayed for the presence of IFN-γ by ELISA. Since the absolute values varied from experiment to experiment, to facilitate comparisons we normalized all values in relation to ConA stimulation. Data are expressed as the ratio between HSP and ConA stimulation multiplied by 100. Values represent means of triplicate samples with SD. *, P = 0.05; **, P < 0.01 with the respective controls.

FIG. 4.

(a) Pf70C- and (b) EB200-specific IgG response elicited in mice primed with live (L) or heat-killed (HK) M. vaccae followed by boosting with Pf70C-EB200 3 weeks later. Immunization and bleeding were done as previously described for BCG. (c) Analysis of Pf70C-specific serum IgG1 and IgG2a isotype levels in mice primed with heat-killed M. vaccae. Serial dilutions of sera from individual mice (three mice per group) were studied. The results from the 1:400 dilutions are presented, with mean OD (A₄₀₅ nm) and SD shown. Statistical analysis showed significant differences between the groups. Background reactivity of the sera with BSA at the corresponding dilution was subtracted. (d) In vitro production of IFN-γ by splenocytes stimulated with Pf70C-EB200 or PPD derived from mice primed with M. vaccae or not primed. Values are the means and SD derived from three spleens. Values represent means of triplicate samples with SD. *, P = 0.05; **, P < 0.01 with the respective controls.

FIG. 5.

(a) ELISA measurement of IgG anti-ML65 and anti-MTB70 antibody response elicited in mice after priming with heat-killed (HK) M. vaccae and boosting with ML65 and MTB70, respectively. Statistical analysis showed significant differences at P values of <0.005 compared with the respective control group by Student's t test (*). (b) ML65- and MTB70-specific IgG1 and IgG2a antibody responses were measured from individual sera, and titers are expressed as the log₁₀ of the reciprocal of the highest dilution giving an OD of 1.0. (c) In vitro IFN-γ production by the splenocytes stimulated with various HSPs from mice primed with heat-killed M. vaccae. Splenocytes of individual mice from each group were stimulated separately in triplicate with 10 μg/ml of each HSP and with 2 μg/ml of ConA (positive control) in 96-well cell culture plates and incubated for 72 h at 37°C in 5% CO₂. Cell supernatants were collected, pooled, and assayed for the presence of IFN-γ by ELISA. Data are expressed as the ratio between HSP and ConA stimulation multiplied by 100. Values represent means of triplicate samples (three spleens) with SD. *, P < 0.005; **, P = 0.07 with the respective controls.

FIG. 6.

Cross-reactivity of total Igs, IgM, and IgG1 antibodies against histone, TNP-BSA, DNA, and collagen III elicited in mice primed or not primed with heat-killed M. vaccae followed by boosting twice with MTB70. Antibody responses were measured from pooled sera of three mice per group. The sera were diluted in consecutive twofold dilutions, starting from 1:20 up to 1:640. ODs from 1:160 dilutions are presented here.

FIG. 7.

Analysis for the presence of renal immune complex deposits of total Igs by using a direct immunofluorescence method. A direct immunofluorescence assay was performed with FITC-conjugated rabbit anti-mouse Igs incubated on a kidney cryostat section from (a) M. vaccae-primed and MTB70 (two times)-boosted mice, (b) HgCl₂-treated SJL mice, and (c) nonimmunized mice. Staining of a glomerulus is seen at 63× magnification. We show here the staining with a 1:40 dilution of the FITC-conjugated rabbit anti-mouse Igs.