Plasma antibodies against heat shock protein 70 correlate with the incidence and severity of asthma in a Chinese population
© Yang et al. 2005
Received: 24 November 2004
Accepted: 14 February 2005
Published: 14 February 2005
The heat shock proteins (Hsps) are induced by stresses such as allergic factors and inflammatory responses in bronchi epithelial cells and therefore may be detectable in patients with asthma. However, the etiologic link between anti-Hsps and asthma (its severity and related inflammatory responses such as interleukin-4 and immunoglobulin E) has not been established. We determined whether antibodies against Hsp60 and Hsp70 were present in patients with asthma and evaluated their associations with risk and severity of asthma.
We determined the levels of anti-Hsp60 and anti-Hsp70 by immunoblot and their associations with risk and symptom severity of asthma in 95 patients with asthma and 99 matched non-symptomatic controls using multivariate logistic regression analysis.
Compared to the controls, asthma patients were more likely to have detectable anti-Hsp60 (17.2% vs 5.1%) and anti-Hsp70 (33.7% vs 8.1%) (p ≤ 0.001). In particular, the presence of anti-Hsp70 was associated with a greater than 2 fold risk for asthma (adjusted OR = 2.21; 95% CI = 1.35~3.59). Furthermore, both anti-Hsp60 and anti-Hsp70 levels were positively correlated with symptom severity (p < 0.05) as well as interleukin-4 and immunoglobulin E (p < 0.05). Individuals with antibodies against anti-Hsp60 and anti-Hsp70 were more likely to have a family history of asthma (p < 0.001) and higher plasma concentrations of total immunoglobulin E (p = 0.001) and interleukin-4 (p < 0.05) than those without antibodies.
These data suggest that anti-Hsp60 and especially anti-Hsp70 correlate with the attacks and severity of asthma. The underlying molecular mechanisms linking antibodies to heat shock proteins and asthma remain to be investigated.
Heat shock proteins (Hsps) are highly conserved proteins inducible in response to a wide variety of stresses (such as exposure to heat) and pathological (viral, bacterial or parasitic infections, and inflammation) or physiological (growth factors, cell differentiation, and hormonal stimulation) stimuli [1, 2]. There are six main Hsp families (i.e., Hsp110, Hsp90, Hsp/Hsc70, Hsp60, Hsp40, and Hsp10-30) categorized on the basis of their apparent molecular masses detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Hsps are involved in various biological functions including 1) intracellular chaperones of naive, aberrantly folded or mutated proteins, 2) cytokines of signal transduction cascades involved in inflammatory response, and 3) cytoprotective agents in response to the aforementioned stress stimuli [1, 3, 4]. In addition, Hsps are also involved in transport of proteins and peptides through cellular compartments, and can bind to endogenous antigenic peptides and transport them to the major histocompatibility complexes [5, 6]. This suggests that Hsps may modulate immune and inflammatory responses and may be involved in the pathogenesis and/or be markers for risk and prognosis of certain diseases including asthma [7–13], given that many of the stress stimuli mentioned above are factors that can induce attacks of asthma.
Asthma is a multifactorial and likely multigenic immune inflammatory disease of the upper airways, arising from complex interactions among environmental and genetic factors [14, 15]. These factors may induce Hsp60 and Hsp70 in bronchi epithelial cells during the development of asthma . Some Hsps present as self-antigens to the immune system, resulting in the production of autoantibodies in patients with inflammatory diseases and immune disorders after infections by bacteria, mycobacteria and Chlamydia [17–19]. Studies have demonstrated that these autoantibodies against Hsps were involved in the pathogenesis and/or prognosis of some diseases [8, 20–23].
Up to now, few studies investigated possible associations of autoantibodies to human Hsps with the severity of asthma. In the present study, we determined the presence of autoantibodies to human Hsp60 and Hsp70 in 193 subjects with (n = 95) and without (n = 99) asthma by immunoblot analysis, and evaluated the associations of these autoantibodies with asthma severity and their correlation with interleukin-4 (IL-4) and immunoglobulin E (IgE) both involved in the development of asthma, by using multivariate logistic regression analyses.
Subjects and groups
Comparison of selected variables between patients with asthma and healthy controls
Patients with asthma (n = 95)
Control subjects (n = 99)
Age (years, mean ± SD)
28.2 ± 15.8
29.1 ± 13.9
Attack once a week or a day (yes/no)
Sign (wheezing) (yes/no)
58.4 ± 18.9
96.5 ± 9.8
IgE (IU/ml, mean ± SD)
486.9 ± 595.5
75.8 ± 124.9
IL-4 (ng/L, mean ± SD)
31.7 ± 17.1
5.1 ± 3.8
Determination of anti-Hsp60 and anti-Hsp70
Detection of plasma IgE and IL-4
Total IgE was measured in plasma by using a fluorescence enzyme immunoassay kit from Bayer Company (Leverkusen, Germany). IL-4 was determined using a commercial enzyme-linked immunosorbent assay kit from OptEIA (Pharmingen, California, U.S.A). Each sample was tested in duplicate by a series of dilutions using a standard provided with the kit.
All the continuous data (e.g., age, FEV1, IGE, IL4) were presented as the mean ± standard deviation (SD) and analysed by the Student's t test. Frequency data (e.g., sex) were analysed by the Chi-square test. The associations were estimated by fitting univariate and multivariate logistic regression models. Statistical inferences were based on a significance level of 0.05. All analyses were two-sided and performed by using the Statistical Package for Social Sciences (SPSS) software (Version, Chicago).
The patient and control groups comprised 56.8% and 64.6% of males, respectively, and had mean ages of 28.2 and 29.1 years, respectively (Table 1). All patients had regular asthma attacks and sign of wheezing, while none of the controls showed any of these signs. Asthma patients had a significantly lower FEV1%Pre than the controls (58.4 vs 96.5, p < 0.001). In addition, the asthma patients had significantly higher concentrations of total IgE and IL-4 than the controls (p < 0.001 for all comparisons).
Presence of anti-Hsp60 and anti-Hsp70 in plasma
Comparison of positive rates of different titers for ant-Hsp60 and anti-Hsp70 in plasma of patients with asthma and healthy controls
Patients with asthma (n = 95)
Control subjects (n = 99)
Association between anti-Hsp60 and anti-Hsp70 with risk for asthma
Multivariate logistic regression analysis of the association between anti-Hsp60 and anti-Hsp70 with risk for asthma
Adjusted Regression coefficient
OR (95% CI)**
Correlation of anti-Hsp60 and anti-Hsp70 with the severity of asthma
Correlation of anti-Hsp70 and anti-Hsp60 with symptom severities
Anti-Hsp70 No. (%)
Anti-Hsp60 No. (%)
Step2: mild persistent
Step3&4 moderate & severe persistent**
Differences in the levels of IgE and IL-4 between asthma patients with positive and negative anti-Hsps
Differences in selected risk factors, IgE, and IL-4 between asthma patients with positive and negative anti-Hsps
Anti-Hsps(+) (n = 37)
Anti-Hsps(-) (n = 58)
Age (years, mean ± SD)
25.6 ± 15.6
29.3 ± 15.7
Family history (yes/no)
IgE (IU/ml, mean ± SD)
758.2 ± 685.3
313.9 ± 458.2
IL-4 (ng/L, mean ± SD)
36.9 ± 17.2
28.5 ± 16.3
Correlation between anti-Hsps, IgE, and IL-4 in 95 asthma patients
The patients included in the present study had frequent asthmatic attacks, with signs of wheezing and higher levels of IgE and IL4 and low levels of FEV1 %Pre that are characteristics of asthma. We found that these asthma patients also had a significantly higher incidence of autoantibodies against combined Hsp60 and Hsp70 than the matched non-asthmatic controls and that, in particular, the presence of anti-Hsp70 was associated with asthma. Furthermore, there was a significant positive correlation between anti-Hsp60 and anti-Hsp70 and symptom severity of asthma. Thus among asthma patients, those who had positive anti-Hsp60 and anti-Hsp70 were more likely to report a family history of asthma and had higher levels of IgE and IL-4 than those without such antibodies. These findings provide evidence to support the hypothesis that the presence of anti-Hsp60 and especially anti-Hsp70 in asthma patients is strongly associated with asthma and the presence of these antibodies may predict symptom severity of asthma and provide new strategies for diagnosis and perhaps treatment of this disease.
Asthma is an immune and inflammatory disease, arising from complex interactions among genetic and environmental factors including bacterial or viral infection [14, 15]. The production of autoantibodies against Hsps may result from genetic factors, infection, denaturation and release of Hsps as a result of cell damage, and the presence of antigen-specific lymphocytes [22, 23].
Hsps are often the target of humoral and T cell-mediated immune responses to infection and may provide a link between the immune response to infection and autoimmunity caused by T lymphocyte cross-reactivity among Hsps of different origins [8, 27, 28]. It remains to be determined whether there is a relationship between the induction of Hsp70 and production of plasma autoantibodies against this Hsp and whether there is a cross-response of induced Hsps and autoantibodies against Hsps before and during the development of asthma. However, there are several lines of evidence that support an association between anti-Hsp60 and anti-Hsp70 and symptom severity in asthma patients. Firstly, as molecular chaperones, Hsps facilitate the synthesis, folding, assembly and intracellular trafficking of many functional proteins [3, 29] and protect cells and organs against different types of damages [30, 31] as observed in transient protection from ischemic injury in whole organs such as heart, brain and kidney [31–34]. Hsp70 has also been suggested to play an autoprotective role in asthma and lung injury [35–37]. Secondly, autoantibodies against Hsps may have significant roles in the pathogenesis and prognosis of diseases. For example, Shinghai et al reported the presence of antibodies against Hsps in patients with autoimmune liver diseases and suggested that the presence of anti-Hsp70 was an indicator for the disease activity of primary biliary cirrhosis . Earlier results from our lab also suggested that the presence of such antibodies might help assess if workers are experiencing abnormal stress within their living and working environment [21–23]. Xu et al and Schett et al have shown that mycobacterial Hsp65 may serve as an antigen to instigate chronic immune responses characteristic of human atherosclerosis. These antibodies were sustained among patients with the most severe degree of underlying atherosclerosis and were demonstrated to predict 5-year mortality [11, 12, 20]. Thirdly, enhanced expression of Hsp70 has been detected in bronchi and alveolar macrophages of patients with asthma and correlated with intrapulmonary eosinophilia, airway inflammation, hyperresponsiveness of bronchi , and severity of the disease [14, 35, 39]. A cross-response of induced plasma and cellular Hsps and autoantibodies against Hsps in human, has been suggested to play a role in the development and prognosis of atherosclerosis [40, 41]. However, it is still unknown whether there is a cross-response between the induction of Hsps in bronchi of patients with asthma and the presence of anti-Hsps and its biological effects.
The development of most immune diseases depends on the cytokines interleukin-2 and interferon-γ produced by type 1 helper T cells (Th1), whereas the development of allergic diseases requires IL-4 and IL-5, both of which are produced by type 2 helper T cells (Th2). The reciprocal down-regulation of Th1 cells by Th2 cytokines raises the possibility that these cytokines are involved in allergy or immunity . IL-4 is one of the first signals for a switch to the synthesis of IgE and IL-4 binds to receptors on B cells to induce and amplify the synthesis of IgE . There is a cross-linking of IgE with allergens to activate a series of response seen in asthma . Epidemiological and clinical observations have linked IgE antibodies to the severity of asthma and the initial and sustained responses of the airways to allergens [45, 46]. At this time, the molecular events that link antibody to Hsp70 to the production of IL-4 and IgE and the interaction among these factors in patients with asthma remain to be investigated.
The present study showed that there was a significant increase in positive rates of antibodies against Hsp60 and Hsp70 in patients with asthma and that the presence of autoantibodies against Hsp70 was associated with the severity of asthma. The presence of anti-Hsp70 associated with a high risk of asthma was also correlated with the family history of asthma and higher levels of total IgE and IL-4 in the patients. These results suggest that anti-Hsp70 correlate with the pathogenesis of asthma, but the precise underlying molecular mechanisms for these interactions remain to be established.
We are particularly grateful to all individuals who voluntarily participated in the study and to the many members of the medical personnel of Hubei College of Traditional Medicine for their generous help in the examination and sampling of subjects. This study was supported by grants from the National Key Basic Research and Development Program (2002CB512905), the National Natural Science Foundation of China (NNSFC 30200227), an exchange Program between the Canadian Institutes of Health Research (CIHR) and the NNSFC (to TW, RMT) and an operating grant from the CIHR (to RMT).
- Morimoto RI, Tissieres A, Georgopoulos C: Progress and perspectives in the biology of heat shock proteins and molecular chaperones. In The Biology of Heat Shock Proteins and Molecular Chaperones. Edited by: Morimoto RI, Tissières A, Georgopoulos C. New York: Cold Spring Harbor Laboratory Press; 1994:1–30.
- Michaud S, Marin R, Tanguay RM: Regulation of heat shock gene induction and expression during Drosophila development. Cell Mol Life Sci 1997, 53:104–113.View ArticlePubMed
- Hightower LE: Heat shock, stress protein, chaperones, and proteotoxicity. Cell 1991, 66:191–197.View ArticlePubMed
- Asea A, Kraeft SK, Kurt-Jones E, Stevenson MA, Chen LB, Finberg R, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000, 6:435–442.View ArticlePubMed
- Singh-Jasuja H, Hilf N, Arnold-Schild D, Schild H: The role of heat shock proteins and their receptors in activation of immune system. Biol Chem 2001, 382:629–636.View ArticlePubMed
- Sato K, Torimoto Y, Tamura Y, Shindo M, Shinzaki H, Hirai K, Kohgo Y: Immunotherapy using heat-shock protein preparations of leukemia cells after syngeneic bone marrow transplantation in mice. Blood 2001, 98:1852–1857.View ArticlePubMed
- Minowada G, Welch WJ: Clinical implications of the stress response. J Clin Invest 1995, 95:3–12.PubMed CentralView ArticlePubMed
- Shingai R, Maeda T, Onishi S, Yamamoto Y: Autoantibody against 70 kD heat-shock protein in patients with autoimmune liver diseases. J Hepatol 1995, 23:382–390.View ArticlePubMed
- Xiao C, Wu T, Ren A, Pan Q, Chen S, Wu F, Li X, Wang W, Hightower LE, Tanguay RM: Basal and inducible levels of Hsp70 in patients with acute heat-induced illness induced during training. Cell Stress Chaperones 2003, 8:86–92.PubMed CentralView ArticlePubMed
- Xiao C, Chen S, Li J, Hai T, Lu Q, Sun E, Wang R, Tanguay RM, Wu T: Association of HSP70 and genotoxic damage in lymphocytes of workers exposed to coke-oven emission. Cell Stress Chaperones 2002, 7:396–402.PubMed CentralView ArticlePubMed
- Xu Q, Willeit J, Marosi M, Kleindienst R, Oberhollenzer F, Kiechl AS, Stulning T, Luef G, Wick G: Association of serum antibodies to heat shock protein 65 with carotid atherosclerosis. Lancet 1993, 341:255–259.View ArticlePubMed
- Xu Q, Kiechl S, Mayr M, Metzler B, Egger G, Oberhollenzer F, Wick G: Association of serum antibodies to heat shock protein 65 with carotid atherosclerosis: clinical significance determined in a follow-up study. Circulation 1999, 100:1169–1174.View ArticlePubMed
- Blachere NE, Srivastava PK: Heat shock protein-based cancer vaccines and related thoughts on immunogenicity of human tumors. Semin Cancer Biol 1995, 6:349–355.View ArticlePubMed
- Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, Maisch S, Carr D, Gerlach F, Bufe A, Lauener RP, Schierl R, Renz H, Nowak D, von Mutius E: Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002, 347:869–877.View ArticlePubMed
- Aron Y, Busson M, Polla BS, Dusser D, Lockhart A, Swierczewski E, Favatier F: Analysis of hsp 70 gene polymorphism in allergic asthma. Allergy 1999, 54:165–170.View ArticlePubMed
- Vignola AM, Chanez P, Polla BS, Vic P, Godard P, Bousquet J: Increased expression of heat shock protein 70 on airway cells in asthma and chronic bronchitis. Am J Respir Cell Mol Biol 1995, 13:683–691.View ArticlePubMed
- Hahn DL, Peeling RW, Dillon E, McDonald R, Saikku P: Serologic markers for Chlamydia pneumoniae in asthma. Ann Allergy Asthma Immunol 2000, 84:227–233.View ArticlePubMed
- Gern JE, Lemanske RF Jr: Infectious triggers of pediatric asthma. Pediatr Clin North Am 2003, 50:555–575.View ArticlePubMed
- Lemanske RF Jr: Is asthma an infectious disease? Thomas A. Neff lecture. Chest 2003, (3 Suppl):385S-390S.
- Schett G, Xu Q, Amberger A, Van Der Zee R, Recheis H, Willeit J, Wick G: Autoantibodies against heat shock protein 65 mediate endothelial cytotoxicity. J Clin Invest 1995, 96:2569–2577.PubMed CentralView ArticlePubMed
- Wu T, Yuan Y, Wu Y, He H, Zhang G, Tanguay RM: Presence of antibodies to heat stress proteins in workers exposed to benzene and in patients with benzene poisoning. Cell Stress Chaperones 1998, 3:161–167.PubMed CentralView ArticlePubMed
- Wu T, Chen S, Sun Y, Xiao C, Wang C, Pan Q, Wang Z, Xie M, Mao Z, Wu Y, Tanguay RM: Presence of antibody against the inducible Hsp71 in patients with acute heat-induced illness. Cell Stress Chaperones 2001, 6:113–120.PubMed CentralView ArticlePubMed
- Wu T, Ma J, Chen S, Sun Y, Xiao C, Gao Y, Wang R, Poudrier J, Dargis M, Currie RW, Tanguay RM: Association of plasma antibodies against the inducible Hsp70 with hypertension and harsh working conditions. Cell Stress Chaperones 2001, 6:394–401.PubMed CentralView ArticlePubMed
- American Thoracic Society: Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987, 136:225–244.View Article
- US Department of Health and Human Sevices: Global initiative for asthma: Global strategy for asthma management and prevention. Bethesda, Maryland: National Institute of Health, National Heart, Lung, and Blood Institute; 2002.
- Tanguay RM, Wu Y, Khandjian EW: Tissue-specific expression of heat shock stress proteins of the mouse in the absence of stress. Dev Genet 1993, 14:112–118.View ArticlePubMed
- Beatty WL, Byrne GI, Morison RP: Morphologic and antigenic characterization of interferon γ-mediated persistent Chlamydia trachomatis infection in vitro. Proc Natl Acad Sci USA 1993, 90:3998–4002.PubMed CentralView ArticlePubMed
- Wick G, Romen M, Amberger A, Metzler B, Mayr M, Falkensammer G, Xu Q: Atherosclerosis, autoimmunity, and vascular-associated lymphoid tissue. FASEB J 1997, 11:1199–1207.PubMed
- Hartl F-U: Molecular chaperones in protein folding. Nature 1996, 381:571–580.View ArticlePubMed
- Angelidis CE, Lazaridis I, Pagoulatos GN: Constitutive expression of heat shock protein 70 in mammalian cells confers thermotolerance. Eur J Biochem 1991, 199:35–39.View ArticlePubMed
- Li GC, Li LY, Liu K, Mak JK, Chen L, Lee WMF: Thermal response of rat fibroblasts stably transfected with the human 70 kDa heat shock protein encoding gene. Proc Natl Acad Sci USA 1991, 88:1681–1685.PubMed CentralView ArticlePubMed
- Plumier JC, Krueger AM, Currie RW, Kontoyiannis D, Kollias G, Pagoulatos GN: Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones 1997, 2:162–167.PubMed CentralView ArticlePubMed
- Plumier JC, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, Pagoulatos GN: Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 1995, 95:1854–1860.PubMed CentralView ArticlePubMed
- Beck FX, Neuhofer W, Müller E: Molecular chaperones in the kidney: Distribution, putative roles, and regulation. Am J Physiol Renal Physiol 2000, 279:F203-F215.PubMed
- Bertorelli G, Bocchino V, Zhou X, Zanini A, Bernini MV, Damia R, Di Comite V, Grima P, Olivieri D: Heat-shock protein 70 upregulation is related to HLD-DR expression in bronchial asthma. Effects of inhaled glucocorticoids. Clin Exp Allergy 1998, 28:551–560.View ArticlePubMed
- Wong HR, Wispe JR: The stress response and the lung. Am J Physiol 1997, 273:L1-L9.PubMed
- Bonay M, Soler P, Riquet M, Battesti JP, Hance AJ, Tazi A: Expression of heat shock proteins in human lung and lung cancers. Am J Respir cell Mol Biol 1994, 10:453–461.View ArticlePubMed
- Tong W, Luo W: Heat shock proteins' mRNA expression in asthma. Respirology 2000, 5:227–230.View ArticlePubMed
- Rha YH, Taube C, Haczku A, Joetham A, Takeda K, Duez C, Siegel M, Aydintug MK, Born WK, Dakhama A, Gelfand EW: Effect of microbial heat shock proteins on airway inflammation and hyperresponsiveness. J Immunol 2002, 169:5300–5307.View ArticlePubMed
- Perschinka H, Mayr M, Millonig G, Mayerl C, Van Der Zee R, Morrison SG, Morrison RP, Xu Q, Wick G: Cross-reactive B-cell epitopes of microbial and human heat shock protein 60/65 in atherosclerosis. Arteroscler Thromb Vas Biol 2003, 23:1060–1065.View Article
- Mandal K, Jahangiri M, Xu Q: Autoimmunity to heat shock proteins in atherosclerosis. Autoimmun Rev 2004, 3:31–37.View ArticlePubMed
- Hopfenspirger MT, Parr SK, Hopp RJ, Townley RG, Agrawal DK: Mycobacterial antigens attenuate late phase response, airway hyperresponsiveness, and bronchoalveolar lavage eosinophilia in a mouse model of bronchial asthma. Int Immunopharmacol 2001, 1:1743–1751.View ArticlePubMed
- Busse WW, Lemanske RF Jr: Asthma. N Engl J Med 2001, 344:350–362.View ArticlePubMed
- Bacharier LB, Jabara H, Gela RS: Molecular mechanisms of immunoglobulin E regulation. Int Arch Allergy Immunol 1998, 115:257–269.View ArticlePubMed
- Burrows B, Martinez FD, Halone M, Barbee RA, Cline MG: Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989, 320:271–277.View ArticlePubMed
- Varner AE, Lemanske RF Jr: The early and late response to allergen. In Asthma and rhinitis. Edited by: Busse WW, Holgate ST. London: Blackwell Science; 2000:1172–1185.View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.