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Ann Thorac Surg 1999;68:1150-1153
© 1999 The Society of Thoracic Surgeons

Original Articles

Heat shock protein suppresses the senescent lung cytokine response to acute endotoxemia

Joseph LoCicero, III, MDa, Xiangjun Xu, MD, PhDa,b, Lihua Zhang, MSa,b

a Section of General Thoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
b Department of Pharmacology, Albert Einstein College of Medicine, New York, New York, USA

Address reprint requests to Dr LoCicero III, Section of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, 110 Francis St, Suite 2C, Boston, MA 02215
e-mail: locicero{at}harvarda.harvard.edu

Presented at the Poster Session of the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–29, 1999.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Previous reports demonstrate that heat shock protein (HSP) can alter the pulmonary inflammatory cascade. We wished to determine if this mechanism is active in the senescent mouse.

Methods. A dose-response and time-response curve for sodium arsenite (SA) induction of HSP was constructed. Eight 25-month-old B6C3F1 mice were given either 1, 2, 4, or 6 mg/kg SA. At 4 hours, the lungs were harvested and assayed for HSP by Western blot. Next, 8 mice were given 4 mg/kg SA and the lungs harvested at either 1, 2, 4, or 6 hours after injection and assayed for HSP. Next, 12 mice were prepared: Half received 4 mg/kg SA and 4 hours later, all received 0.5 mg/kg lipopolysaccharide (LPS). After 4 hours, lungs were harvested and Interleukin-1ß mRNA was assayed by Northern blot and semiquantified by densitometry.

Results. The optimum SA dose was determined to be 4 mg/kg. The maximum HSP production was at 4 hours. Mice receiving LPS only showed a marked increase (3-fold) in IL-1 message compared with the mice pretreated with SA.

Conclusions. These data suggest that in the senescent as in the mature mouse lung, HSP downregulates the inflammatory cascade in response to LPS.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Old lungs are less resistant than young lungs to various types of stress. Acute lung injury is one of the clinically common stresses to lungs, and several studies have shown that old lungs are more easily injured by acute lung injury than are young lungs [1, 2]. Lipopolysaccharide (LPS) is the commonly used endotoxin that produces local inflammation of the lung as well as the systemic toxicity of gram-negative infection. Interleukin-1ß (IL-1ß) is one of the earliest produced and most important LPS-induced proinflammatory cytokines [3]. IL-1ß is produced by resident lung and circulating macrophages. Locally accentuated activation of IL-1ß plays an important role in inflammation and the regulation of acute phase genes [4]. The inhibition of expression of IL-1ß could downregulate acute lung injury. Although IL-1ß is vital to the process of acute lung injury, relatively little is known regarding its regulation.

Heat shock protein (HSP) is a useful probe with which to dissect the role of IL-1ß in the pathogenesis of inflammatory and immunologically mediated diseases [5, 6]. When organisms are exposed to sufficiently severe heat shock or stress conditions, the majority die. However if prior to this lethal heat shock, they undergo a mild heat treatment, a considerable proportion of them will survive [7]. Sodium arsenite (SA), an arsenic compound, has been used to induce HSP in different cell lines. It can induce HSP without an obligate increase in the body temperature [8, 9].


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
This project was approved by the Animal Care and Use Committee of Harvard Medical School and The Animal Care and Use Committee of Beth Israel Deaconess Medical Center. The experiments conformed to all of the regulations concerning humane treatment of laboratory animals.

IL-1ß mRNA expression in old lungs
Three groups of B6C3F1 mice (6 in each group) received 0.5 mg/kg intraperitoneal LPS (026;b6, DIFCO Laboratories, Detroit, MI): young (2-month-old), mature (8-month-old), and old (25-month-old) acquired from National Institute of Aging, Bethesda, MD. Four hours later, the mice were killed and the lungs were frozen and individually homogenized, fractionated on an agarose gel, and IL-1ß mRNA detected by hybridization to an IL-1ß mRNA cDNA probe using standard Northern blot techniques. These results were compared with ß-actin gene as reference. A fourth group of mice without LPS assayed as controls. Blotting was semiquantified by normalized integrated optical densitometry and expressed as relative percentages.

HSP-72 dose-response curve
Sodium arsenite (Sigma Chemical, St. Louis, MO) was diluted in saline and injected peritoneally. Progressive doses (1.0 to 6.0 mg/kg) were given for generation of HSP dose- and time-course curves. Eight 25-month-old mice (2 in each group) received increasing doses of sodium arsenite into peritoneal. After 4 hours, these animals were killed with pentobarbital overdose. The chest was then opened, and the lungs were removed and immediately frozen in liquid nitrogen. The samples were stored at -85°C until Western analysis was performed.

HSP-72 time-course curve
Eight 25-month-old mice (2 in each group), to provide a time-response curve of HSP-72 expression in the lungs (the lowest dose of sodium arsenite which produced the highest amount of HSP expression with minimal toxicity [optimal dose] was chosen), received sodium arsenite 4 mg/kg and were killed at different time-points (1, 2, 4, and 6 hours) after injection. The lungs were processed for determination of HSP-72 by standard Western blot analysis.

HSP blocking experiment
Twelve mice were used for this experiment. Six controls received LPS without HSP induction. The other 6 mice received the optimal dose given at the optimal time prior to the intraperitoneal injection of LPS. At 4 hours after the LPS injections, all animals were killed, and the lungs were frozen and individually homogenized, fractionated on an agarose gel, and IL-1ß mRNA detected by hybridization to an IL-1ß mRNA cDNA probe using Northern blot technique. These results were compared with ß-actin gene as reference. Blotting was semiquantified by normalized integrated optical densitometry and expressed as relative percentages.

Statistical analysis
The data are expressed as mean ± standard deviation and groups compared using Student’s t test corrected for small sample size. Data were considered statistically significant for p less than 0.05.


   Results
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 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Inflammatory response to LPS challenge
We measured the response of the senescent mouse to a septic challenge in order to determine the appropriate time needed to develop a significant level of IL-1ß mRNA response. There was a progressive increase in the amount of IL-1ß message. The IL-1ß mRNA response in the senescent mice responded at 1 to 2 hours, and then rose and was sustained at a significantly higher level with a slight decrease at 6 hours. Figure 1 presents the normalized densitometry values in graphical form: 1 hour, 5 ± 1.2%; 2 hours, 11 ± 1.25%; 3 hours, 48 ± 2.1%; 4 hours, 52 ± 1.1%; 5 hours, 49 ± 1.3%; 6 hours, 47 ± 2.1% (p < 0.05: 4 hours compared with 1 and 2 hours).



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  		Fig 1. The normalized integrated optical densities of the Northern blots of the senescent mouse IL-1ß response to LPS challenge. Note the increased response beginning around 2.5 hours.

 
Figure 2 shows graphically the results of the normalized densitometry of the Northern blots of IL-1ß mRNA dynamics in young and old murine lung tissue during acute endotoxemia at the 4-hour time point. The baseline levels of IL-1ß mRNA were nearly undetectable in all unstimulated animals. After 4 hours of stimulation with LPS, the levels were: 7 ± 1.2% for ß-actin; 18 ± 2.8% in 2-month-old mice; 26 ± 3.2% in 8-month-old mice; and 50 ± 3.3% in 25-month-old mice (p < 0.001 compared with 2-month-old; p < 0.05 compared with 8-month-old mice).



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  		Fig 2. The normalized integrated optical densities of the Northern blots of mouse IL-1ß response to LPS challenge at the 4-hour time point. The level of the 25-month-old mice was high compared with the mice aged 2 months (p < 0.001) and 8 months (p < 0.05).

 
HSP induction
We next determined the appropriate dose and time interval in which to induce the HSP prior to the start of the septic challenge. Figure 3 shows representative densitometry plots of the Western blots of HSP expression during stimulation with SA. Increasing doses of SA produced increased expression of HSP-72. Mice receiving 8 mg/kg demonstrated a high toxicity. The optimum dose was determined to be 4 mg/kg. Another 8 mice (2 for each group) were given this dose and the time interval for development of HSP observed. There was an increase in HSP-72 expression in the lungs within 3 to 4 hours, then a slow decrease.



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  		Fig 3. The normalized integrated optical densities of the Western blots for dose and time. Units on the abscissa are mg/kg for the dose ranging study and hours for the time study, using 4 mg/kg as the dose.

 
HSP suppression of IL-1ß mRNA
Figure 4 shows normalized densitometry of the Northern blots of the suppression of IL-1ß mRNA expression in old lungs to LPS after HSP induction. Mice receiving LPS only showed a 3-fold increase in IL-1ß mRNA message compared with mice pretreated with SA: ß-actin, 5 ± 1.1%; IL-1ß mRNA without SA, 57 ± 2.2%; IL-1ß mRNA with SA, 12 ± 1.3% (p < 0.01).



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  		Fig 4. The normalized integrated optical densities of the Northern blots with and without HSP suppression. Note the marked diminution in IL-1ß mRNA with HSP induction (p < 0.001).

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
One of the characterizations of physiological aging is altered responses to stress. In many cases, baseline levels of physiological parameters do not change with aging, but when the systems are stressed, we observe an age-related decrement in survival to stress. Incidence of gram-negative bacteremia is higher in elderly adults, and more elderly people are hospitalized after exposure to gram-negative challenge. It has been suggested that elderly people are at greater risk for the development of gram-negative bacterial infections than younger individuals [1, 2].

In our experiment, we found that when challenged with LPS, old lungs have highly elevated levels of IL-1ß mRNA expression compared with young and mature groups of mice. These results suggest a progressive exaggeration with aging of the pulmonary inflammatory response to an equipotent septic challenge. Although a specific mechanism for the process of aging has not been fully elucidated, approaches now permit the examination of the effect of aging on the response to stress at the molecular level [10, 11]. During the evolution of inflammation, the host responds with the production of a number of cytokines which produce inflammation [12]. Of particular relevance to aging is the suggestion that a pharmacologic or genetic agent might be found to bind to denatured or abnormal proteins produced by stress and aid in their elimination [13–15].

As the major endotoxin in gram-negative infections, LPS can stimulate the expression of a large number of cytokines that orchestrate inflammation. LPS-initiated expression of host-derived mediators rather than LPS per se may be responsible for the proinflammatory effects [16, 17]. Interleukin-1ß is one of the most important LPS-induced proinflammatory cytokines. IL-1ß mRNA is expressed after the intraperitoneal injection of LPS as measured by IL-1ß mRNA levels in whole organ preparations [3]. The kinetics of IL-1ß mRNA expression was studied in mice of various ages including elderly (near end-of-life) mice. We observed a progressive increase in the amount of IL-1ß message with increasing age. In particular, the IL-1ß mRNA response in senescent mice began at 1 to 2 hours, then rose and was sustained at a significantly higher level than baseline (Fig 1). This suggests that it would be very important to develop a method to protect against the acute injury in senescent lung.

The exposure of cells to a variety of metabolic or environmental stresses results in the preferential synthesis of a group of highly conserved proteins referred to as the HSPs. These proteins are generally presumed to increase the ability of cells to recover from the toxic effects of physiological stresses. When organisms are exposed to sufficiently severe stress conditions, the majority die. However, if prior to this severe stress, they undergo a mild heat treatment or heat stress induction, a considerable proportion of them survive.

Heat shock response appears to protect the cell from injury and also enhance cellular recovery from physiological stresses [17]. Because elderly people are less likely to develop elevated body temperature, there is a potential that the protective effect of heating observed in these studies was not directly due to the synthesis of HSP but to some nonspecific effects of heating such as glucocorticoid release or synthesis of prostaglandin E2 [18]. SA has been reported to induce HSP in various organs. SA was used to induce HSP in the lungs and seemed to provide protection against acute lung injury without the confounding effects of heat. We found that increasing expression of HSP peaked at 4 hours after SA injection (4 mg/kg; Fig 3). At this dose, the mice did not demonstrate signs of toxicity such as lethargy or anorexia.

We have demonstrated that pretreatment of SA before exposure of old lungs to LPS resulted in divergent changes in the expression of IL-1ß mRNA. The IL-1ß mRNA although abundantly expressed in the presence of LPS was inhibited when HSP was induced by SA (Fig 4). Since HSP is known to inhibit the expression of many proteins [19], we also examined the effects on the expression of a constitutively expressed RNA polymerase II gene, ß-actin. We found that SA induced HSP inhibits expression of IL-1ß without alteration in the expression of the ß-actin. Therefore, the inhibition of IL-1ß mRNA is not the consequence of a general inhibition of gene expression. These results suggest that HSP may have important antiinflammatory effects.

The physiological function of IL-1ß mRNA and IL-1ß mRNA regulation during whole body stress and infection is very important. This is the first demonstration of HSP as a repressor in modulation of proinflammatory IL-1ß mRNA expression in the senescent lung. The antiinflammatory potential of HSP in modulating pulmonary inflammatory process is tied directly to its alteration of neutrophil function and the suppression of cytokines derived from the alveolar macrophage. The inhibition of alveolar macrophage derived IL-1ß mRNA may attenuate the influx of inflammatory cells into the distal airways and reduce the subsequent injury in the old lung.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Hoffman-Goetz L., Keir R. Fever and survival in aged mice after endotoxin challenge. J Gerontol 1985;40:15-22.[Abstract/Free Full Text]
  2. Maiello M., Boeri D., Sampietro L., Pronzato M.A., Odetti P., Marinari U.M. Basal synthesis of heat shock protein-70 increases with age in rat kidneys. Gerontology 1998;44:15-20.[Medline]
  3. Van der Poll T., de Waal Malefyt R., Coyle S.M., Lowry S.F. Antiinflammatory cytokine responses during clinical sepsis and experimental endotoxemia. J Infect Dis 1997;175:118-122.[Medline]
  4. Minowada G., Welch W.J. Clinical implications of the stress response. J Clin Invest 1995;95:3-12.
  5. Bonay M.P., Soler P., Riquet M., Battesti J.P., Hance A.J., Tazi A. Expression of heat shock proteins in human lung and lung cancers. Am J Respir Cell Mol Biol 1994;10:453-461.[Abstract]
  6. Angeletti B., Pascale E., Verna R., Passarelli F., Butler R.H., D’Ambrosio E. Differential expression of heat shock protein (HSP-70) mRNAs in rat cells. Exp Cell Res 1996;227:160-164.[Medline]
  7. Wong H.R., Mannix R.J., Rusnak J.M., et al. The heat shock response attenuates lipopolysaccharide-mediated apoptosis in cultured sheep pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 1996;15:745-751.[Abstract]
  8. Villar J., Ribeiro S.P., Mullen J.B., Kuliszewski M., Post M., Slutsky A.S. Sodium arsenite reduces mortality rate and organ damage in a sepsis-induced acute lung injury model. Crit Care Med 1994;22:914-922.[Medline]
  9. Ribeiro S.P., Villar J., Downey G.P., Edelson J.D., Slutsky A.S. Sodium arsenite induces heat shock protein-72 kilodalton expression in the lungs and protects rats against sepsis. Critical Care Med 1994;22:922-929.[Medline]
  10. Chirgwin J.J., Przbyla A.E., MacDonald R.J., Rutter W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979;18:5294-5299.[Medline]
  11. Carlyle W.C., Toher C.A., Vandervelde J.R., McDonald K.M., Homans D.C., Cohn J.N. Changes in ß-actin mRNA expression in remodeling. J Mol Cell Cardiol 1996;28:53-63.[Medline]
  12. Ho Y.S., Dey M.S., Crapo J.D. Antioxidant enzyme expression in rat lungs during hyperoxia. Am J Physiol 1996;270:L810-L818.[Abstract/Free Full Text]
  13. Matuschak G.M., Munoz C.F., Johanns C.A., Rahman R., Lechner A.J. Upregulation of postbacteremic TNF alpha and IL-1 alpha gene expression by alveolar hypoxia/reoxygenation in perfused rat lungs. Am J Respir Crit Care Med 1998;157:629-637.[Abstract/Free Full Text]
  14. Van der Poll T., Keogh C.V., Burman W.A., Kopf M., Lowry S.F. Passive immunization against tumor necrosis factor alpha impairs host defense during pneumococcal pneumonia in mice. Am J Respir Crit Care Med 1997;155:603-608.[Abstract]
  15. Sawa T., Corry D.B., Gropper M.A., Ohara M., Kurahashi K., Wiener-Kornish J.P. IL-10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia. J Immunol 1997;159:2858-2866.[Abstract]
  16. Tremblay L., Valenza F., Ribeiro S.P., Li J., Slutzky A.S. Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997;99:944-952.[Medline]
  17. Shenkar R., Abraham E. Hemorrhage induces rapid in vivo activation of CREB and NFkappaB in murine intraparenchymal lung mononuclear cells. Am J Respir Cell Mol Biol 1997;16:145-152.[Abstract]
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  19. Goral J., Mathews H.L., Clancy J., Jr Expression of 70kDa heat shock protein during acute graft versus host disease. Clin Immunol Immunopathol 1998;86:252-258.[Medline]



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