Mycobacterial 65-kD Heat Shock Protein Research Articles

Antibody to mycobacterial 65-kD heat shock protein in commercial antisera.

Inhibition ELISA and immunoblotting were used to examine the antigenic cross-reactivity claimed to exist between mycobacterial 65-kD heat shock protein (hsp65) and human lactoferrin. Commercially available anti-lactoferrin antibodies produced using either Freund's complete (FCA) or Freund's incomplete adjuvant were tested for binding to recombinant mycobacterial hsp65. Both antibody preparations showed reactivity with hsp65, this being greater with the antibody produced using FCA. However, we found no evidence of a cross-reaction. Lactoferrin failed to inhibit anti-hsp65 reactivity, while hsp65 itself did. Affinity purified anti-lactoferrin antibody showed no reaction with hsp65 by ELISA or immunoblotting. These data suggest that commercial anti-lactoferrin preparations are contaminated with antibodies to hsp65. A commercial anti-albumin antibody also bound to hsp65 in ELISA, so this may be a more general phenomenon.

Mycobacterial 65-kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells.

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65-kD heat shock protein (hsp) on their cell surface to alpha beta and gamma delta T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65-kD hsp. However, it is not known whether 65-kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65-kD hsp may directly activate THP-1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose-dependent and abolished by heat-induced proteolysis. Subsequently, THP-1 cells secreted IL-6 and IL-8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65-kD hsp may be important in the host immune response and in the development of antigen-specific T cell-mediated immunity.

Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning.

The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC16); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.

Real-time Quantitative PCR in the Diagnosis of Tuberculosis in Formalin-fixed Paraffin-embedded Pleural Tissue in Patients From a High HIV Endemic Area

The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

Mycobacterial Hsp65‐IgG–expressing tolerogenic B cells confer protection against adjuvant‐induced arthritis in Lewis rats

Tolerization of T cells directed against a target autoantigen is a desired goal of experimental approaches for the treatment of autoimmune diseases, and novel and improved methods of tolerance induction are continuously being sought. Because most traditional methods of tolerance induction using soluble antigen are effective in the prevention of autoimmunity but fail to control established disease, this study was carried out to explore an innovative tolerogenic approach for the treatment of ongoing disease, using the rat adjuvant-induced arthritis (AIA) model of human rheumatoid arthritis. Lewis (RT.1(l)) rats were injected subcutaneously with heat-killed Mycobacterium tuberculosis H37Ra to induce AIA. Before or after AIA induction, Lewis rats were treated intraperitoneally (IP) with tolerogenic B cells expressing a fusion construct of mycobacterial 65-kd heat-shock protein (Hsp65) and IgG heavy-chain. For comparison, control rats were treated IP with ovalbumin (OVA)-IgG-expressing B cells or soluble mycobacterial Hsp65, and the effects on AIA were observed. We also tested the immune response to mycobacterial Hsp65 in B cell-tolerized rats. Administration of tolerogenic mycobacterial Hsp65-expressing B cells as well as soluble mycobacterial Hsp65, but not OVA-expressing B cells, resulted in a significant decrease in the severity of subsequent AIA. However, in rats with established disease, only the B cell regimen of mycobacterial Hsp65, but not the soluble antigen, suppressed ongoing AIA. Mycobacterial Hsp65-IgG-expressing B cells can successfully attenuate the progression of AIA. This study introduces a promising approach for the treatment of arthritis that should be further explored.

Inhibition of adjuvant‐induced arthritis by DNA vaccination with the 70‐kd or the 90‐kd human heat‐shock protein: Immune cross‐regulation with the 60‐kd heat‐shock protein

Adjuvant arthritis can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt). The mycobacterial 65-kd heat-shock protein (Hsp65) is targeted by arthritogenic T cells. However, Hsp65 and the mycobacterial 71-kd heat-shock protein are also recognized by T cells that can down-regulate adjuvant-induced arthritis (AIA). We have recently demonstrated that vaccination with human Hsp60 DNA inhibits AIA. The present study was undertaken to analyze the role of the T cell responses to self HSP molecules other than Hsp60 in the control of AIA. Lewis rats were immunized with DNA vaccines coding for human Hsp70 or Hsp90 (Hsp70 plasmid [pHsp70] or pHsp90), and AIA was induced. The T cell response to Mt, Hsp60, Hsp70, and Hsp90 (proliferation and cytokine release) was studied, and the T cell response to Hsp60 was mapped with overlapping peptides. The Hsp70 or Hsp90 DNA vaccines shifted the arthritogenic T cell response from a Th1 to a Th2/3 phenotype and inhibited AIA. We detected immune crosstalk between Hsp70/90 and Hsp60: both the Hsp70 and Hsp90 DNA vaccines induced Hsp60-specific T cell responses. Similarly, DNA vaccination with Hsp60 induced Hsp70-specific T cell immunity. Epitope mapping studies revealed that Hsp60-specific T cells induced by pHsp70 vaccination reacted with known regulatory Hsp60 epitopes. T cell immunity to Hsp70 and to Hsp90, like Hsp60-specific immunity, can modulate the arthritogenic response in AIA. In addition, our results suggest that the regulatory mechanisms induced by Hsp60, Hsp70, and Hsp90 are reinforced by an immune network that connects their reactivities.

Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis.

Lewis rats are susceptible to several forms of experimental arthritis-induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II-restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell-mediated regulation of inflammation.

Activation of Synovial Fluid T Lymphocytes by 60‐Kd Heat‐Shock Proteins in Patients with Inflammatory synovitis

Synovial fluid lymphocytes from patients with rheumatoid arthritis and with other forms of inflammatory synovitis demonstrate enhanced proliferative responses to Mycobacterium tuberculosis antigens, in particular, the 65-kd heat-shock protein. There is a high degree of homology between the human and the mycobacterial 60-kd family of heat-shock proteins. These studies were performed to determine if the enhanced response to the mycobacterial 65-kd heat-shock protein was due to cross-reactivity of an immune response generated against the human homolog. These studies were performed by in vitro culture of isolated synovial fluid mononuclear cells with crude and purified antigens. The synovial fluid lymphocytes of a majority of patients with rheumatoid arthritis recognized the mycobacterial 65-kd heat-shock protein, as evidenced by T cell proliferation. In contrast, only 18% of all samples tested responded to a highly purified recombinant human 60-kd heat-shock protein. With only one exception, proliferative responses to the mycobacterial antigen were stronger than those to the human homolog. The proliferative responses generated against mycobacterial 65-kd heat-shock proteins from different sources were highly correlated. The findings suggest that the enhanced proliferative response to the mycobacterial 65-kd heat-shock protein noted in most patients with rheumatoid arthritis and other forms of inflammatory synovitis is not due to cross-reactivity of an immune response directed against the human heat-shock protein.

Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones.

Adjuvant arthritis in rats is induced by a T cell clone specific for amino acids 180-188 of the mycobacterial 65-kD heat-shock protein, and synovial T cell responses to this same Ag have been noted in human arthritis. We have isolated 65-kD Ag-specific T cell clones from synovial fluid mononuclear cells of a patient with acute arthritis, which, unlike the corresponding PBMC, showed a marked proliferative response to the 65-kD Ag. Using synthetic peptides corresponding to the whole sequence of the 65-kD Ag, all the clones were shown to recognize an epitope present in the first NH2-terminal peptide (amino acids 1-15), with no response to the adjacent peptide (amino acids 6-22) or to any other peptide. The complete dominance of this epitope in the response to the 65-kD Ag was shown by documenting responses to the peptide in PBMC obtained after recovery from the arthritis. This epitope, like that recognized by the rat arthritogenic T cell clone, is in a portion of the 65-kD sequence that is not conserved between bacteria and eukaryotes, so that in this case, joint inflammation could not be attributed to bacteria-induced T cell clones cross-reacting with the self 65-kD Ag.

A mycobacterial 65-kD heat shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic.

A recombinant (r)65-kD protein from Mycobacterium leprae, at levels far in excess of those present in whole mycobacteria, was unable to induce arthritis. Even when combined with a synthetic adjuvant, CP20961, to mimic the peptidoglycan adjuvant component of the mycobacterial cell wall, the r65-kD protein failed to induce arthritis. Pretreatment with as little as 1 microgram r65-kD protein protected rats against arthritis induced by M. tuberculosis, but this r65-kD protein was markedly less able to protect against arthritis induced by the synthetic adjuvant, CP20961, or type II collagen. The r65-kD protein appears, therefore, to produce an antigen-specific protection against arthritis induced by bacterial cell walls containing the 65-kD protein. Such protection can be overcome, however, by arthritogenic T lymphocytes, suggesting that protection occurs by preventing clonal proliferation of autoreactive T lymphocytes that are induced by the adjuvant properties of mycobacterial cell walls. How the r65-kD protein abrogates this particular adjuvant activity, and the nature of the arthritogenic self antigen(s), remain to be elucidated.

The mycobacterial 65 kD heat-shock protein and autoimmune arthritis

Arthritis - induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis. Recent findings from the analysis of immunological reactivity to the 65-kD in rheumatoid arthritis patients seem to support such a role for the mycobacterial 65-kD heat-shock protein in human disease.

Protection against streptococcal cell wall-induced arthritis by pretreatment with the 65-kD mycobacterial heat shock protein.

We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW. In such protected rats, SCW-specific T cell responses were suppressed, as compared with responses in arthritic rats. Pretreatment with 65-kD protein had no effect on the production of antibodies against SCW, on a nonspecific inflammatory reaction (zymosan-induced arthritis), or on general cellular immunity in vivo (delayed type hypersensitivity reaction to a nonrelated protein antigen). Furthermore, the protection against SCW arthritis was transferable by splenic T cells to naive recipients. Our data show that pretreatment with the 65-kD mycobacterial heat shock protein protects rats against a subsequent bacterium-induced arthritis. This protection is immunologically specific and resides in the lymphoid cell population.