Heat-Shock Protein Synthesis in Chicken Macrophages: Influence of In Vivo and In Vitro Heat Shock, Lead Acetate, and Lipopolysaccharide1 L. MILLER and M. A. QURESHI 2 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608
1992 Poultry Science 71:988-598
INTRODUCTION Exposure to elevated environmental temperatures and to other forms of cellu-
Received for publication September 13, 1991. Accepted for publication February 5, 1992. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Services nor criticism of similar ones not mentioned. To whom correspondence should be addressed.
lar injury can induce in whole organisms and cells a physiological reaction classically termed the heat-shock (HS) response (Schlesinger et ah, 1982). The HS response in all species examined is characterized by heightened transcription and preferential translation of mRNA belonging to several evolutionary conserved families of genes, resulting in the increased synthesis of HS proteins (HSP) (Lindquist, 1986; Lindquist and Craig, 1988). These stress proteins are classified according to their approximate
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ABSTRACT Synthesis of heat-shock proteins (HSP) in chicken macrophages, in response to thermal and nonthermal stressors, was determined. Cornell Kstrain 6-wk-old White Leghorn females were injected with Sephadex and approximately 42 h later subjected to elevated temperatures in order to achieve a core body temperature (CBT) of 44 C. Peritoneal macrophages were isolated at 30 and 60 min after heat treatment. A parallel group of chickens, maintained at the normal CBT of 41C, was used as controls and peritoneal macrophages were isolated after 60 min of treatment. For in vitro study of HSP response, cells of a chicken macrophage cell line (MQ-NCSU) were subjected to 45 C ambient temperature to produce heat shock (HS, thermal stress), lipopolysaccharide (LPS, 15 ug), and lead acetate (nonthermal stress) exposure for varying time periods. The HSP profiles of macrophages following various treatments were determined by one- and two-dimensional gel electrophoresis. The results showed that macrophages isolated from the 44 C CBT group synthesized HSP90, HSP70, HSP23, and a heat-inducible P32 protein. This HSP synthesis profile was similar to the HSP expression by MQ-NCSU cells exposed in vitro to 45 C conditions. Exposure to MQ-NCSU cells to lead acetate induced the same four proteins previously expressed by macrophages after in vivo or in vitro heat treatment. Two-dimensional analysis of lysates from cells treated with LPS, HS, or LPS plus HS treatments revealed a doublet protein molecule (70a and 70b) with identical molecular mass of 70 kDa. However, the pi value (isoelectric point) of 70b was higher (5.1) than that of 70a, which, along with HSP90 and HSP23, focused more toward the acidic side with a pi value of less than 4.6. The present study is the first to report pi profiles of chicken macrophage HSP. The in vitro and in vivo studies suggest that chicken macrophages respond to thermal and nonthermal stressors by producing similar kinds of "stress proteins". (Key words: heat stock, lead acetate, lipopolysaccharide, chicken macrophage, stress proteins)
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
The induction of increased HSP synthesis has been observed in avian immunological cell types, including chicken lymphocytes and lymphoblastoid (MSB) cells cultured under HS conditions (Morimoto and Fodor, 1984; Banerji et al, 1986). Furthermore, the induction of HSP synthesis in chicken fibroblast and chicken embryonic cells occurs after in vitro exposure to HS, inhibitors of energy metabolism, amino acid analogues, chelators, and several heavy metals such as copper, cadmium, zinc, and mercury (Kelley and Schlesinger, 1978; Levinson et al, 1978, 1980; Johnston et al, 1980; Kelley et al, 1980). Studies on temperature regulation in various avian species, including chickens, indicate that birds have the ability to regulate their core body temperature (CBT) within a constant level of 40.5 to 41 C within a wide range of ambient temperatures (Hillman et al, 1985). California quail (Lovhortyx californicus) have developed special adaptations of thermal regulation and water metabolism that allow them to tolerate CBT of 44 C for 1 h or
even 46 C for 15 min imposed by exposure to high ambient temperatures (Hudson and Brush, 1964; Brush, 1965). Dean and Atkinson (1985) observed that the red blood cells of hyperthermic Japanese quail (Coturnix coturnix japonica) with CBT between 42.0 and 43.2 C for 80 min synthesized enhanced levels of several HSP, including HSP23, HSP70, and HSP90. These investigators suggested that the rapid synthesis of these HSP, within the tolerable hyperthermic range of quail, may be an important physiological mechanism allowing for cell survival at extreme environmental temperatures or thermal stress. Chicken peritoneal macrophages exposed to 45 C in vitro (thermal stress) have been shown to produce three major HSP species, namely HSP23, HSP70, a n d HSP90 (Miller and Qureshi, 1992a). Further studies have shown that both cell lineages (e.g., lymphoid versus myeloid) and avian species respond differently in terms of HSP induction during thermal stress (Miller and Qureshi, 1992b). In addition, macrophage effector functions such as nonspecific phagocytosis (Miller and Qureshi, 1992a) and tumoricidal potential (Miller and Qureshi, 1992c) is significantly reduced within an HS environment. In the present study, chickens were exposed to elevated temperatures to achieve 44 C CBT. Protein profiles of HS macrophages isolated from these chickens were then examined. In addition, the question whether nonthermal stressors such as lipopolysaccharide (LPS) and lead acetate (PbAc) can also induce a stress response in macrophages was investigated. Furthermore, the present study is the first to describe pi values of chicken macrophage HSP, where p i is the isoelectric point, defined as the p H at which the net positive and negative charge on a protein is zero.
MATERIALS AND METHODS Chickens Cornell K-strain (B15Bt5) 6-wk-old female White Leghorn chickens (200 to 300 g body weight) were employed in in vivo HS
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molecular mass into three major protein families: HSP90, HSP70, HSP60, and a fourth small HSP family that is composed of proteins ranging from approximately 15 to 30 kDa (Hemmingsen et al, 1988; Lindquist and Craig, 1988). Recently, investigations of the HS response have focused on the role of HSP in inflammation and the expression of stress proteins in various mammalian immunological cell types, including polymorphonuclear leukocytes, basophils, B and T lymphocytes, and monocytes (Eid et al, 1987; Maridonneuu-Parini and Polla, 1987; Polla et al, 1987; Ferris et al, 1988; Spector et al, 1989; Ciavarra and Simeone, 1990; Koller and K6nig, 1990). Polla et al (1988) observed that human monocytes preexposed to mild HS conditions synthesized HSP, which partially protected these cells from exogenous ^CVmcurced cell death. Further studies by Clerget and Polla (1990) determined that the induction of HSP synthesis during macrophage erythrophagocytosis might result from their endogenous generation of oxygenfree radicals in the presence of hemoglobin-derived iron.
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experiments. The chickens were reared in raised wire cages. Feed and water were available for ad libitum consumption. They were fed a corn-soybean starter diet composed of 17.4% crude protein and 2,900 kcal of ME/kg, which met the requirement for essential nutrients (National Research Council, 1984).
Macrophage Cell Line
Peritoneal Macrophages Sephadex-elicited chicken peritoneal exudate cells were harvested as previously described (Trembicki et ah, 1984). Viable cell concentration of peritoneal exudate cells was determined by trypan blue dye exclusion (Phillips, 1973) and adjusted to 5 x 105 cells/mL. To obtain adherent macrophage monolayers, 1 mL of this cell suspension was placed in each well of a 24-well tissue culture plate. 3 The plates were then incubated at 41 C in a humidified incubator with 5% CO2 for 1 h. The nonadherent cells were then removed by repeatedly washing with sterile .85% saline. The washed culture dishes were then replenished with RPMI1640 growth medium supplemented with 10% heat-inactivated fetal calf serum, 100 U / m L of penicillin, 50 u g / m L of streptomycin, and 2 ug fungizone (CM) 4 and reincubated until subjected to their respective treatments. This procedure yields an
3 Costar, Cambridge, MA 04923. •kabco, Grand Island, NY 14072. Lab-Line Instruments, Inc., Melrose Park, IL 60160. "Yellow Springs Instruments, Inc., Yellow Springs, OH 45387. 7 Difco Laboratories, Detroit, MI 48233.
MQ-NCSU Cultures The MQ-NCSU cells were harvested from flasks by gentle agitation, washed twice in Leibovitz-McCoy's complete medium, and the viable cell concentration as determined by trypan blue dye exclusion was adjusted to 5 x 10 5 cells/mL. To obtain adherent MQ-NCSU cell monolayers, 1 mL of this cell suspension was plated in each well of a 24-well tissue culture plate. 5 The plates were then incubated at 41 C in a humidified incubator with 5% CO2. After 1 h, the medium was removed, the cultures replenished with 1 mL of fresh medium, and subjected to their respective treatments.
In Vivo Heat-Shock Treatment After 42 h of Sephadex injection, six chickens per treatment group were placed in a thermostatically controlled incubator 5 maintained at 41 or 44 C and their CBT were maintained at 41 C for 60 min or until CBT reached 44 C (= 15 min), at which time CBT was maintained at 44 C for 30 or 60 min. The CBT of all birds was constantly monitored using an electronic thermometer probe 6 inserted 5 cm into the cloaca of each bird. Immediately after the application of each thermal treatment, the birds were euthanatized by electrocution, peritoneal exudate cells collected, and macrophage monolayers established for radiolabeling.
Lipopolysaccharide, Lead Acetate, and In Vitro Heat-Shock Treatment of MQ-NCSU Cells The MQ-NCSU cells were exposed to 41 C control (CON) or 45 C HS conditions for 1 h or to 15 ug of LPS (Escherichia coli 055:B5, Westphal) 7 at 41 or 45 C for 9 h in a humidified incubator with 5% CO2. Additionally, MQ-NCSU cells were subjected to 1,000 \iM sodium acetate 3 ; 0, 10, 100, and 1,000 uM PbAc 3 for 3 and 24 h at 41 C in 5% C02-humidified chambers. Following LPS, acetate, PbAc, and HS treatments, the MQNCSU cultures were processed for radiolabeling.
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A newly developed chicken mononuclear cell line, MQ-NCSU (Qureshi et ah, 1990), was employed in these studies. These cells are phagocytic, secrete tumoricidal factor, and exhibit morphological and cell surface antigen characteristics unique to mononuclear phagocytic lineage cells. The MQ-NCSU cells were grown in LeibovitzMcCoy's complete medium (Qureshi et ah, 1990) at 41 C with 5% CO2 in a humidified chamber.
adherent cell preparation consisting of greater than 95% macrophages (Qureshi et ah, 1986).
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
Radiolabeling Macrophages
of Peritoneal and MQ-NCSU Cells
Protein
ICN Biomedicals, Inc., Costa Mesa, CA 92626. Bio-Rad Laboratories, Richmond, CA 94804. DuPont, NEN Research Products, Boston, MA 02118. n K o d a k , Rochester, NY 14650. 9
10
CL in v i t r o HS Con 60m
PM in v i v o HS Con 30m 60m
Analysis
The protein profiles of the peritoneal macrophages and the MQ-NCSU cells exposed to CON, HS, and PbAc treatments were determined by one-dimensional gel electrophoresis of the equal amounts of lysates using a 4% stacking and 10% resolving polyacrylamide gels in the presence of SDS according to the method of Laemmli (1970). The electrophoresis was performed at 10 mA until the tracking dye (bromophenol blue) reached the bottom of the slabs. Additionally, two-dimensional analysis of equal amounts of proteins in the cell lysates of the LPS- and HS-treated MQNCSU cells were performed using the twodimensional gel electrophoresis method according to CFarrell (1975). Isoelectric focusing of samples in the first-dimensional gels were conducted with ampholines 10 (pH range 3 to 10 and 5 to 7). The seconddimensional polyacrylamide gel slabs consisted of a 4% stacking and 10% resolving gel. In all cases, isoelectric focusing markers 1 0 were run in parallel in first-
8
dimensional gels and molecular weight markers 1 0 in the second-dimensional gels. Completed gels containing separated radiolabeled proteins were processed for fluorography according to the NEN autography enhancer protocol, 10 dried in a gel dryer, 9 and exposed to X-ray film 11 with intensifying screens 10 for appropriate times at -70 C. The autoradiographs were developed and subjected to visual examination of the expressed bands. Electrophoretic analysis of two replications of each treatment sample was conducted in two separate experiments.
FIGURE 1. Protein profile of MQ-NCSU cells after in vitro heat-shock (HS) treatment and chicken peritoneal macrophages (PM) isolated from hyperthermic or HS birds. The mononuclear cell line MQNCSU (CL) was exposed in vitro for a period of 1 h to 41 C control (Con) or 45 C HS [60 min (m)] conditions. Sephadex-elicited PM were isolated from hyperthermic chickens subjected to 44 C HS treatment for a period of 30 or 60 m. The Con Sephadexelicited PM were isolated from chickens maintained at a normal physiological temperature of 41 C. The HSP and heat-inducible P32 proteins are indicated with corresponding molecular mass. Actin is indicated as a reference protein.
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After exposure to their respective treatments, the peritoneal macrophages and MQ-NCSU cell cultures were washed with sterile PBS (.6 M, pH 7.4) and replenished with 1 mL of methionine-free RPMI-16404 (pre-equilibrated to 41 C) supplemented with 10% fetal calf serum and antibiotics (100 U / m L penicillin and 50 (ig/mL streptomycin) 4 containing 100 uCi of Trans[ 35 S]labeled methionine 8 with a specific activity of 1,156 Ci/mmoL. After a 1-h labeling period at 41 C, all culture medium from the culture wells was removed and the cells were lysed directly in the wells with 100 jxL of lysing buffer (9 M u r e a , .1 M dithiothreitol, and 2.8% nonidet P-40).9 The cell lysates were stored at -70 C until analysis.
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RESULTS Three major HSP, HSP23, HSP70, and HSP90 were synthesized at elevated levels in Sephadex-elicited peritoneal macrophages collected from chickens that were maintained at a CBT of 44 C (HS condition) for 30 min or 60 min (Figure 1). The increased synthesis of a heat-inducible 32 kDa (P32) protein was also induced by this in vivo HS treatment. Peritoneal macrophages from chickens with CBT maintained at 41 C CON levels did not express these four HSP species. In order to compare the HS response of in vivo HS macrophages with the expression of HSP in macrophages HS in vitro, monocytic MQ-NCSU cell line, cultures were exposed to 41 or 45 C for 1 h. The MQ-NCSU cells subjected to in vitro HS conditions (45 C) expressed all three HSP and the P32 observed in their in vivo counterparts (Figure 1). The protein profiles of MQ-NCSU cells were examined after CON treatment (41 C) with either 1,000 uM acetate (acetate control) or 0, 10, 100, and 1,000 uM of
Con *4h
Ac 24h
10uM 3h
24 h
100uM 3h
24h
1000uM 3h
24h
HSP70-
P32-
FIGURE 2. Protein profile of MQ-NCSU cells after lead acetate (PbAc) treatment. The MQ-NCSU cells were exposed to 10 \lM and 1,000 \iM of PbAc for a period of 3 and 24 h at 41 C. Untreated control (Con) and acetate control (Ac) cells were cultured at 41 C for 24 h without lead. The HSP and heat-inducible P32 proteins indicated with corresponding molecular mass. Actin is indicated as a reference protein.
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PbAc. The MQ-NCSU cells treated with 100 \xM or 1,000 \xM of PbAc for 3 h at 41 C expressed similar HSP23, HSP70, and HSP90, and P32 proteins (Figure 2) as observed in the MQ-NCSU cells exposed to HS conditions (Figure 1). This pattern of HSP expression continued over the 24-h time period tested (Figure 2). The cells treated with 10 \xM PbAc only induced HSP90 synthesis. Exposure of the MQ-NCSU cells to 1,000 ]\M acetate for 24 h did not induce an altered protein pattern when compared with controls cultured at 41 C for 24 h. To further analyze the protein profiles of macrophages in response to thermal (HS) and nonthermal (LPS) stressors, cell lysates were subjected to two-dimensional p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (Figures 3, 4, and 5). Comparisons of twodimensional protein profiles show that two proteins (or possible break down product of one single protein) with a common molecular mass of 70 kDa (70a and 70b) and distinct pi of 5.1 (70b) and less than 4.6 (70a), respectively, are synthesized at enhanced levels by MQ-NCSU
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
DISCUSSION When cultured cells or whole animals are exposed to elevated temperatures or a
multitude of other stressors, they respond by producing a group of proteins called HS or stress proteins (Lindquist, 1986; Lindquist and Craig, 1988). This HS response has been observed in all organisms from bacteria to yeasts, plants, and animals, including cells and tissues isolated from birds (Schlesinger et ah, 1982; Morimoto and Fodor, 1984; Banerji et ah, 1986). In general, it is believed that the HS response, which is characterized by a rapid and transient change in gene expression at both the level of transcription and translation, might serve to protect an organism's cells from lethal damage imposed by heat or some other hostile environmental stressor (Welch, 1990). The present study has shown that Sephadex-elicited peritoneal macrophages
HS pH 9.6
7.5 I
4.6 I
.90
•
*
««Ac
r 23.
FIGURE 3. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 60 min at 45 C heat-shock (HS) conditions. Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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cells exposed to HS, LPS, and combined HS and LPS treatments (Figures 3, 4, and 5) as compared with the untreated CON (Figure 6). Additionally, the enhanced synthesis of 23-kDa and 90-kDa proteins with a pi value of less than 4.6 was also observed in MQ-NCSU cells following exposure to each of these treatments. The isoelectric focusing markers utilized in the present study unfortunately did not allow for the exact assessment of the pi values of the 23-kDa and 90-kDa proteins. The MQNCSU cells exposed to 41 C CON conditions and without LPS treatment did not express HSP (Figure 6).
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A comparison of HSP expression after thermal or nonthermal exposures reveals that macrophages are capable of responding to these stressors in similar fashion by synthesizing HSP or "stress protein". The exposure of MQ-NCSU cells to nonthermal stressors such as PbAc induced increased synthesis of HSP23, HSP70, HSP90, and P32 (Figure 2), resulting in a profile similar to the observed HSP profiles of macrophages after HS (Figure 1). This pattern of HSP expression continues over a 24-h period (Figure 2). The expression and intensity (as observed by visual examination) of expressed proteins was related to the dose of PbAc. Exposure to 10 \xM PbAc induced detectable expression of only HSP90 (Figure 2). The pattern of HSP expression in MQ-NCSU cells follow-
LPS 4.6 I
7.5 i
pH 9.6
70b
#
.90
70E
*
*
»
* ^
Ac
23; V
m
FIGURE 4. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 9 h at 41 C in the presence of 15 ug of Upopolysaccharide (LPS). Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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isolated from hyperthermic chickens synthesize increased levels of HSP. The normal pattern of protein synthesis in peritoneal macrophage from CON birds with a CBT of 41 C is greatly altered, as observed in their HS counterparts by the increased synthesis of HSP23, HSP70, and HSP90 along with a heat-inducible P32 protein (Figure 1). This pattern of HSP expression was observed in peritoneal macrophages isolated from chickens subjected to 30 or 60 min hyperthermiainducing conditions. Additionally, this in vivo HS response by macrophages is similar to that induced in MQ-NCSU cells (a cell line representing cells of mononuclear phagocytic system lineage) (Qureshi et ah, 1990) exposed in vitro to HS (45 C) conditions (Figure 1).
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
(Figures 3, 4, and 5). This doublet molecule has not been observed in the authors' previously reported one-dimensional gel electrophoretic analysis of the induction of HSP in the chicken macrophage (Miller and Qureshi, 1992a,b). Utilizing onedimensional analysis (Miller and Qureshi, 1992c) demonstrated the enhanced synthesis of a heat-inducible P32 in HS- and LPStreated macrophages. A unique P120 was observed in macrophage treated only with LPS. The two-dimensional gel electrophoretic separations used in the present study did not permit the focusing of these two stress proteins. However, the t w o dimensional protein profiles of thermal (HS) and nonthermal (LPS) treated MQNCSU cells with elevated synthesis of HSP23, HSP90, and two HSP70 stress
HS+LPS pH
9.6 I
7.5 I
4.6
^90
^Mk ^W^
23.
FIGURE 5. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 9 h at 45 C heat-shock (HS) conditions in the presence of 15 \lg of lipopolysaccharide (LPS). Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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ing PbAc treatment differs from observations in rat fibroblasts and kidney epithelial cells, which expressed only a single 32-kDa metal-induced protein after 3 h exposure to 1,000 uM of lead glutamate (Shelton et al, 1986). However, these investigators suggest that this 32-kDa protein may be related to the 35-kDa stress protein induced in the chick embryo cells by kethoxal bis (thiosemicarbazone), copper, cadmium, zinc, mercury, and sodium arsenite (Levinson et al, 1978, 1980; Johnston et al, 1980). Two-dimensional electrophoretic separation of lysates from HS, LPS, and combined HS-treated and LPS-treated MQ-NCSU cells show the expression of a doublet protein molecule 70a and 70b with a common molecular mass of 70 kDa
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Con
pH
9.6 I
7.5
4.6 I
*
Ac
FIGURE 6. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 60 min at 41 C control (Con) conditions. Actin (Ac) is indicated as a reference protein.
proteins indicate that common families of HSP are expressed in avian macrophage exposed to hostile environmental conditions. The present study is the first to describe pi values of HSP in chicken macrophages. The pi for HSP70b is approximately 5.1 whereas HSP70a, HSP90, and HSP23 are more acidic, with pi values less than 4.6 (Figures 3, 4, and 5). Previous studies have described acidic pi values of 5.4 for HSP26 and pi ranging from 5.2 to 6.95 for HSP70 and HSP90 in HS quail red blood cells (Morimoto and Fodor, 1984; Dean and Atkinson, 1985). The present study, therefore, suggests that avian macrophages are sensitive to changes in their microenvironment both in vivo and in vitro. They respond by producing HS or stress proteins. Furthermore, as previously report-
ed, macrophages under HS conditions exhibit suppressed phagocytic potential (Miller and Qureshi, 1992a) as well as reduced synthesis of tumoricidal factor (Miller and Qureshi, 1992c). However, this suppression seems to be transient, because the phagocytic potential of macrophages returns to control levels after heat stress is removed. It is therefore speculated that the HS response may act as a physiological mechanism that protects macrophages from thermal and nonthermal damage and allows them to maintain their role in the immune functions under the most hostile conditions. ACKNOWLEDGMENTS The authors thank R. Hunter for excellent technical assistance and M. Robinson
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4*
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and C. Smith for typing the manuscript. The research reported in this publication was funded by North Carolina Agricultural Research Services, Project Number NC06175.
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Qureshi, M. A., L. Miller, H. S. Lillehoj, and M. D. Ficken, 1990. Establishment and characterization of a chicken mononuclear cell line. Vet. Immunol. Immunopathol. 26:237-250. Schlesinger, M. J., P. M. JCelly, G. Aliperti, and C. Malfer, 1982. Properties of three major heatshock proteins and their antibodies. Pages 243-250 in: Heat Shock from Bacteria to Man. M. J. Schlesinger, M. Ashbumes, and A. Tissieres, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shelton, K. R., J. M. Todd, and P. M. Egle, 1986. The induction of stress-related proteins by lead. J. Biol. Chem. 261:1935-1940. Spector, N. L., A. S. Freedman, G. Freeman, J. Segil, J.
F. Whitman, W. J. Welch, and L. M. Nadler, 1989. Activation primes human B lymphocytes to respond to heat shock. J. Exp. Med. 170: 1763-1768. Trembicki, K. A., M. A. Qureshi, and R. R. Dietert, 1984. Avian peritoneal exudate cells: A comparison of stimulation protocols. Dev. Comp. Immunol. 8:395-402. Welch, W. J., 1990. The mammalian stress response: Cell physiology and biochemistry of stress proteins. Pages 223-278 in: Stress Proteins in Biology and Medicine. R. Morimoto, A. Tissieres, and C. Georgopolus, ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Downloaded from http://ps.oxfordjournals.org/ at New York University on April 23, 2015
1992 Poultry Science 71:988-598
INTRODUCTION Exposure to elevated environmental temperatures and to other forms of cellu-
Received for publication September 13, 1991. Accepted for publication February 5, 1992. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Services nor criticism of similar ones not mentioned. To whom correspondence should be addressed.
lar injury can induce in whole organisms and cells a physiological reaction classically termed the heat-shock (HS) response (Schlesinger et ah, 1982). The HS response in all species examined is characterized by heightened transcription and preferential translation of mRNA belonging to several evolutionary conserved families of genes, resulting in the increased synthesis of HS proteins (HSP) (Lindquist, 1986; Lindquist and Craig, 1988). These stress proteins are classified according to their approximate
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ABSTRACT Synthesis of heat-shock proteins (HSP) in chicken macrophages, in response to thermal and nonthermal stressors, was determined. Cornell Kstrain 6-wk-old White Leghorn females were injected with Sephadex and approximately 42 h later subjected to elevated temperatures in order to achieve a core body temperature (CBT) of 44 C. Peritoneal macrophages were isolated at 30 and 60 min after heat treatment. A parallel group of chickens, maintained at the normal CBT of 41C, was used as controls and peritoneal macrophages were isolated after 60 min of treatment. For in vitro study of HSP response, cells of a chicken macrophage cell line (MQ-NCSU) were subjected to 45 C ambient temperature to produce heat shock (HS, thermal stress), lipopolysaccharide (LPS, 15 ug), and lead acetate (nonthermal stress) exposure for varying time periods. The HSP profiles of macrophages following various treatments were determined by one- and two-dimensional gel electrophoresis. The results showed that macrophages isolated from the 44 C CBT group synthesized HSP90, HSP70, HSP23, and a heat-inducible P32 protein. This HSP synthesis profile was similar to the HSP expression by MQ-NCSU cells exposed in vitro to 45 C conditions. Exposure to MQ-NCSU cells to lead acetate induced the same four proteins previously expressed by macrophages after in vivo or in vitro heat treatment. Two-dimensional analysis of lysates from cells treated with LPS, HS, or LPS plus HS treatments revealed a doublet protein molecule (70a and 70b) with identical molecular mass of 70 kDa. However, the pi value (isoelectric point) of 70b was higher (5.1) than that of 70a, which, along with HSP90 and HSP23, focused more toward the acidic side with a pi value of less than 4.6. The present study is the first to report pi profiles of chicken macrophage HSP. The in vitro and in vivo studies suggest that chicken macrophages respond to thermal and nonthermal stressors by producing similar kinds of "stress proteins". (Key words: heat stock, lead acetate, lipopolysaccharide, chicken macrophage, stress proteins)
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
The induction of increased HSP synthesis has been observed in avian immunological cell types, including chicken lymphocytes and lymphoblastoid (MSB) cells cultured under HS conditions (Morimoto and Fodor, 1984; Banerji et al, 1986). Furthermore, the induction of HSP synthesis in chicken fibroblast and chicken embryonic cells occurs after in vitro exposure to HS, inhibitors of energy metabolism, amino acid analogues, chelators, and several heavy metals such as copper, cadmium, zinc, and mercury (Kelley and Schlesinger, 1978; Levinson et al, 1978, 1980; Johnston et al, 1980; Kelley et al, 1980). Studies on temperature regulation in various avian species, including chickens, indicate that birds have the ability to regulate their core body temperature (CBT) within a constant level of 40.5 to 41 C within a wide range of ambient temperatures (Hillman et al, 1985). California quail (Lovhortyx californicus) have developed special adaptations of thermal regulation and water metabolism that allow them to tolerate CBT of 44 C for 1 h or
even 46 C for 15 min imposed by exposure to high ambient temperatures (Hudson and Brush, 1964; Brush, 1965). Dean and Atkinson (1985) observed that the red blood cells of hyperthermic Japanese quail (Coturnix coturnix japonica) with CBT between 42.0 and 43.2 C for 80 min synthesized enhanced levels of several HSP, including HSP23, HSP70, and HSP90. These investigators suggested that the rapid synthesis of these HSP, within the tolerable hyperthermic range of quail, may be an important physiological mechanism allowing for cell survival at extreme environmental temperatures or thermal stress. Chicken peritoneal macrophages exposed to 45 C in vitro (thermal stress) have been shown to produce three major HSP species, namely HSP23, HSP70, a n d HSP90 (Miller and Qureshi, 1992a). Further studies have shown that both cell lineages (e.g., lymphoid versus myeloid) and avian species respond differently in terms of HSP induction during thermal stress (Miller and Qureshi, 1992b). In addition, macrophage effector functions such as nonspecific phagocytosis (Miller and Qureshi, 1992a) and tumoricidal potential (Miller and Qureshi, 1992c) is significantly reduced within an HS environment. In the present study, chickens were exposed to elevated temperatures to achieve 44 C CBT. Protein profiles of HS macrophages isolated from these chickens were then examined. In addition, the question whether nonthermal stressors such as lipopolysaccharide (LPS) and lead acetate (PbAc) can also induce a stress response in macrophages was investigated. Furthermore, the present study is the first to describe pi values of chicken macrophage HSP, where p i is the isoelectric point, defined as the p H at which the net positive and negative charge on a protein is zero.
MATERIALS AND METHODS Chickens Cornell K-strain (B15Bt5) 6-wk-old female White Leghorn chickens (200 to 300 g body weight) were employed in in vivo HS
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molecular mass into three major protein families: HSP90, HSP70, HSP60, and a fourth small HSP family that is composed of proteins ranging from approximately 15 to 30 kDa (Hemmingsen et al, 1988; Lindquist and Craig, 1988). Recently, investigations of the HS response have focused on the role of HSP in inflammation and the expression of stress proteins in various mammalian immunological cell types, including polymorphonuclear leukocytes, basophils, B and T lymphocytes, and monocytes (Eid et al, 1987; Maridonneuu-Parini and Polla, 1987; Polla et al, 1987; Ferris et al, 1988; Spector et al, 1989; Ciavarra and Simeone, 1990; Koller and K6nig, 1990). Polla et al (1988) observed that human monocytes preexposed to mild HS conditions synthesized HSP, which partially protected these cells from exogenous ^CVmcurced cell death. Further studies by Clerget and Polla (1990) determined that the induction of HSP synthesis during macrophage erythrophagocytosis might result from their endogenous generation of oxygenfree radicals in the presence of hemoglobin-derived iron.
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experiments. The chickens were reared in raised wire cages. Feed and water were available for ad libitum consumption. They were fed a corn-soybean starter diet composed of 17.4% crude protein and 2,900 kcal of ME/kg, which met the requirement for essential nutrients (National Research Council, 1984).
Macrophage Cell Line
Peritoneal Macrophages Sephadex-elicited chicken peritoneal exudate cells were harvested as previously described (Trembicki et ah, 1984). Viable cell concentration of peritoneal exudate cells was determined by trypan blue dye exclusion (Phillips, 1973) and adjusted to 5 x 105 cells/mL. To obtain adherent macrophage monolayers, 1 mL of this cell suspension was placed in each well of a 24-well tissue culture plate. 3 The plates were then incubated at 41 C in a humidified incubator with 5% CO2 for 1 h. The nonadherent cells were then removed by repeatedly washing with sterile .85% saline. The washed culture dishes were then replenished with RPMI1640 growth medium supplemented with 10% heat-inactivated fetal calf serum, 100 U / m L of penicillin, 50 u g / m L of streptomycin, and 2 ug fungizone (CM) 4 and reincubated until subjected to their respective treatments. This procedure yields an
3 Costar, Cambridge, MA 04923. •kabco, Grand Island, NY 14072. Lab-Line Instruments, Inc., Melrose Park, IL 60160. "Yellow Springs Instruments, Inc., Yellow Springs, OH 45387. 7 Difco Laboratories, Detroit, MI 48233.
MQ-NCSU Cultures The MQ-NCSU cells were harvested from flasks by gentle agitation, washed twice in Leibovitz-McCoy's complete medium, and the viable cell concentration as determined by trypan blue dye exclusion was adjusted to 5 x 10 5 cells/mL. To obtain adherent MQ-NCSU cell monolayers, 1 mL of this cell suspension was plated in each well of a 24-well tissue culture plate. 5 The plates were then incubated at 41 C in a humidified incubator with 5% CO2. After 1 h, the medium was removed, the cultures replenished with 1 mL of fresh medium, and subjected to their respective treatments.
In Vivo Heat-Shock Treatment After 42 h of Sephadex injection, six chickens per treatment group were placed in a thermostatically controlled incubator 5 maintained at 41 or 44 C and their CBT were maintained at 41 C for 60 min or until CBT reached 44 C (= 15 min), at which time CBT was maintained at 44 C for 30 or 60 min. The CBT of all birds was constantly monitored using an electronic thermometer probe 6 inserted 5 cm into the cloaca of each bird. Immediately after the application of each thermal treatment, the birds were euthanatized by electrocution, peritoneal exudate cells collected, and macrophage monolayers established for radiolabeling.
Lipopolysaccharide, Lead Acetate, and In Vitro Heat-Shock Treatment of MQ-NCSU Cells The MQ-NCSU cells were exposed to 41 C control (CON) or 45 C HS conditions for 1 h or to 15 ug of LPS (Escherichia coli 055:B5, Westphal) 7 at 41 or 45 C for 9 h in a humidified incubator with 5% CO2. Additionally, MQ-NCSU cells were subjected to 1,000 \iM sodium acetate 3 ; 0, 10, 100, and 1,000 uM PbAc 3 for 3 and 24 h at 41 C in 5% C02-humidified chambers. Following LPS, acetate, PbAc, and HS treatments, the MQNCSU cultures were processed for radiolabeling.
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A newly developed chicken mononuclear cell line, MQ-NCSU (Qureshi et ah, 1990), was employed in these studies. These cells are phagocytic, secrete tumoricidal factor, and exhibit morphological and cell surface antigen characteristics unique to mononuclear phagocytic lineage cells. The MQ-NCSU cells were grown in LeibovitzMcCoy's complete medium (Qureshi et ah, 1990) at 41 C with 5% CO2 in a humidified chamber.
adherent cell preparation consisting of greater than 95% macrophages (Qureshi et ah, 1986).
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
Radiolabeling Macrophages
of Peritoneal and MQ-NCSU Cells
Protein
ICN Biomedicals, Inc., Costa Mesa, CA 92626. Bio-Rad Laboratories, Richmond, CA 94804. DuPont, NEN Research Products, Boston, MA 02118. n K o d a k , Rochester, NY 14650. 9
10
CL in v i t r o HS Con 60m
PM in v i v o HS Con 30m 60m
Analysis
The protein profiles of the peritoneal macrophages and the MQ-NCSU cells exposed to CON, HS, and PbAc treatments were determined by one-dimensional gel electrophoresis of the equal amounts of lysates using a 4% stacking and 10% resolving polyacrylamide gels in the presence of SDS according to the method of Laemmli (1970). The electrophoresis was performed at 10 mA until the tracking dye (bromophenol blue) reached the bottom of the slabs. Additionally, two-dimensional analysis of equal amounts of proteins in the cell lysates of the LPS- and HS-treated MQNCSU cells were performed using the twodimensional gel electrophoresis method according to CFarrell (1975). Isoelectric focusing of samples in the first-dimensional gels were conducted with ampholines 10 (pH range 3 to 10 and 5 to 7). The seconddimensional polyacrylamide gel slabs consisted of a 4% stacking and 10% resolving gel. In all cases, isoelectric focusing markers 1 0 were run in parallel in first-
8
dimensional gels and molecular weight markers 1 0 in the second-dimensional gels. Completed gels containing separated radiolabeled proteins were processed for fluorography according to the NEN autography enhancer protocol, 10 dried in a gel dryer, 9 and exposed to X-ray film 11 with intensifying screens 10 for appropriate times at -70 C. The autoradiographs were developed and subjected to visual examination of the expressed bands. Electrophoretic analysis of two replications of each treatment sample was conducted in two separate experiments.
FIGURE 1. Protein profile of MQ-NCSU cells after in vitro heat-shock (HS) treatment and chicken peritoneal macrophages (PM) isolated from hyperthermic or HS birds. The mononuclear cell line MQNCSU (CL) was exposed in vitro for a period of 1 h to 41 C control (Con) or 45 C HS [60 min (m)] conditions. Sephadex-elicited PM were isolated from hyperthermic chickens subjected to 44 C HS treatment for a period of 30 or 60 m. The Con Sephadexelicited PM were isolated from chickens maintained at a normal physiological temperature of 41 C. The HSP and heat-inducible P32 proteins are indicated with corresponding molecular mass. Actin is indicated as a reference protein.
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After exposure to their respective treatments, the peritoneal macrophages and MQ-NCSU cell cultures were washed with sterile PBS (.6 M, pH 7.4) and replenished with 1 mL of methionine-free RPMI-16404 (pre-equilibrated to 41 C) supplemented with 10% fetal calf serum and antibiotics (100 U / m L penicillin and 50 (ig/mL streptomycin) 4 containing 100 uCi of Trans[ 35 S]labeled methionine 8 with a specific activity of 1,156 Ci/mmoL. After a 1-h labeling period at 41 C, all culture medium from the culture wells was removed and the cells were lysed directly in the wells with 100 jxL of lysing buffer (9 M u r e a , .1 M dithiothreitol, and 2.8% nonidet P-40).9 The cell lysates were stored at -70 C until analysis.
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RESULTS Three major HSP, HSP23, HSP70, and HSP90 were synthesized at elevated levels in Sephadex-elicited peritoneal macrophages collected from chickens that were maintained at a CBT of 44 C (HS condition) for 30 min or 60 min (Figure 1). The increased synthesis of a heat-inducible 32 kDa (P32) protein was also induced by this in vivo HS treatment. Peritoneal macrophages from chickens with CBT maintained at 41 C CON levels did not express these four HSP species. In order to compare the HS response of in vivo HS macrophages with the expression of HSP in macrophages HS in vitro, monocytic MQ-NCSU cell line, cultures were exposed to 41 or 45 C for 1 h. The MQ-NCSU cells subjected to in vitro HS conditions (45 C) expressed all three HSP and the P32 observed in their in vivo counterparts (Figure 1). The protein profiles of MQ-NCSU cells were examined after CON treatment (41 C) with either 1,000 uM acetate (acetate control) or 0, 10, 100, and 1,000 uM of
Con *4h
Ac 24h
10uM 3h
24 h
100uM 3h
24h
1000uM 3h
24h
HSP70-
P32-
FIGURE 2. Protein profile of MQ-NCSU cells after lead acetate (PbAc) treatment. The MQ-NCSU cells were exposed to 10 \lM and 1,000 \iM of PbAc for a period of 3 and 24 h at 41 C. Untreated control (Con) and acetate control (Ac) cells were cultured at 41 C for 24 h without lead. The HSP and heat-inducible P32 proteins indicated with corresponding molecular mass. Actin is indicated as a reference protein.
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PbAc. The MQ-NCSU cells treated with 100 \xM or 1,000 \xM of PbAc for 3 h at 41 C expressed similar HSP23, HSP70, and HSP90, and P32 proteins (Figure 2) as observed in the MQ-NCSU cells exposed to HS conditions (Figure 1). This pattern of HSP expression continued over the 24-h time period tested (Figure 2). The cells treated with 10 \xM PbAc only induced HSP90 synthesis. Exposure of the MQ-NCSU cells to 1,000 ]\M acetate for 24 h did not induce an altered protein pattern when compared with controls cultured at 41 C for 24 h. To further analyze the protein profiles of macrophages in response to thermal (HS) and nonthermal (LPS) stressors, cell lysates were subjected to two-dimensional p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (Figures 3, 4, and 5). Comparisons of twodimensional protein profiles show that two proteins (or possible break down product of one single protein) with a common molecular mass of 70 kDa (70a and 70b) and distinct pi of 5.1 (70b) and less than 4.6 (70a), respectively, are synthesized at enhanced levels by MQ-NCSU
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
DISCUSSION When cultured cells or whole animals are exposed to elevated temperatures or a
multitude of other stressors, they respond by producing a group of proteins called HS or stress proteins (Lindquist, 1986; Lindquist and Craig, 1988). This HS response has been observed in all organisms from bacteria to yeasts, plants, and animals, including cells and tissues isolated from birds (Schlesinger et ah, 1982; Morimoto and Fodor, 1984; Banerji et ah, 1986). In general, it is believed that the HS response, which is characterized by a rapid and transient change in gene expression at both the level of transcription and translation, might serve to protect an organism's cells from lethal damage imposed by heat or some other hostile environmental stressor (Welch, 1990). The present study has shown that Sephadex-elicited peritoneal macrophages
HS pH 9.6
7.5 I
4.6 I
.90
•
*
««Ac
r 23.
FIGURE 3. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 60 min at 45 C heat-shock (HS) conditions. Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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cells exposed to HS, LPS, and combined HS and LPS treatments (Figures 3, 4, and 5) as compared with the untreated CON (Figure 6). Additionally, the enhanced synthesis of 23-kDa and 90-kDa proteins with a pi value of less than 4.6 was also observed in MQ-NCSU cells following exposure to each of these treatments. The isoelectric focusing markers utilized in the present study unfortunately did not allow for the exact assessment of the pi values of the 23-kDa and 90-kDa proteins. The MQNCSU cells exposed to 41 C CON conditions and without LPS treatment did not express HSP (Figure 6).
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A comparison of HSP expression after thermal or nonthermal exposures reveals that macrophages are capable of responding to these stressors in similar fashion by synthesizing HSP or "stress protein". The exposure of MQ-NCSU cells to nonthermal stressors such as PbAc induced increased synthesis of HSP23, HSP70, HSP90, and P32 (Figure 2), resulting in a profile similar to the observed HSP profiles of macrophages after HS (Figure 1). This pattern of HSP expression continues over a 24-h period (Figure 2). The expression and intensity (as observed by visual examination) of expressed proteins was related to the dose of PbAc. Exposure to 10 \xM PbAc induced detectable expression of only HSP90 (Figure 2). The pattern of HSP expression in MQ-NCSU cells follow-
LPS 4.6 I
7.5 i
pH 9.6
70b
#
.90
70E
*
*
»
* ^
Ac
23; V
m
FIGURE 4. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 9 h at 41 C in the presence of 15 ug of Upopolysaccharide (LPS). Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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isolated from hyperthermic chickens synthesize increased levels of HSP. The normal pattern of protein synthesis in peritoneal macrophage from CON birds with a CBT of 41 C is greatly altered, as observed in their HS counterparts by the increased synthesis of HSP23, HSP70, and HSP90 along with a heat-inducible P32 protein (Figure 1). This pattern of HSP expression was observed in peritoneal macrophages isolated from chickens subjected to 30 or 60 min hyperthermiainducing conditions. Additionally, this in vivo HS response by macrophages is similar to that induced in MQ-NCSU cells (a cell line representing cells of mononuclear phagocytic system lineage) (Qureshi et ah, 1990) exposed in vitro to HS (45 C) conditions (Figure 1).
HEAT-SHOCK PROTEIN SYNTHESIS IN CHICKEN MACROPHAGE
(Figures 3, 4, and 5). This doublet molecule has not been observed in the authors' previously reported one-dimensional gel electrophoretic analysis of the induction of HSP in the chicken macrophage (Miller and Qureshi, 1992a,b). Utilizing onedimensional analysis (Miller and Qureshi, 1992c) demonstrated the enhanced synthesis of a heat-inducible P32 in HS- and LPStreated macrophages. A unique P120 was observed in macrophage treated only with LPS. The two-dimensional gel electrophoretic separations used in the present study did not permit the focusing of these two stress proteins. However, the t w o dimensional protein profiles of thermal (HS) and nonthermal (LPS) treated MQNCSU cells with elevated synthesis of HSP23, HSP90, and two HSP70 stress
HS+LPS pH
9.6 I
7.5 I
4.6
^90
^Mk ^W^
23.
FIGURE 5. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 9 h at 45 C heat-shock (HS) conditions in the presence of 15 \lg of lipopolysaccharide (LPS). Heat-shock proteins are indicated with arrows along with the corresponding molecular mass (e.g., 23, 70a, 70b, and 90). Actin (Ac) is indicated as a reference protein.
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ing PbAc treatment differs from observations in rat fibroblasts and kidney epithelial cells, which expressed only a single 32-kDa metal-induced protein after 3 h exposure to 1,000 uM of lead glutamate (Shelton et al, 1986). However, these investigators suggest that this 32-kDa protein may be related to the 35-kDa stress protein induced in the chick embryo cells by kethoxal bis (thiosemicarbazone), copper, cadmium, zinc, mercury, and sodium arsenite (Levinson et al, 1978, 1980; Johnston et al, 1980). Two-dimensional electrophoretic separation of lysates from HS, LPS, and combined HS-treated and LPS-treated MQ-NCSU cells show the expression of a doublet protein molecule 70a and 70b with a common molecular mass of 70 kDa
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Con
pH
9.6 I
7.5
4.6 I
*
Ac
FIGURE 6. Fluorogram of two-dimensional electrophoretic separation of the proteins from lysates of MQNCSU macrophages cultured for 60 min at 41 C control (Con) conditions. Actin (Ac) is indicated as a reference protein.
proteins indicate that common families of HSP are expressed in avian macrophage exposed to hostile environmental conditions. The present study is the first to describe pi values of HSP in chicken macrophages. The pi for HSP70b is approximately 5.1 whereas HSP70a, HSP90, and HSP23 are more acidic, with pi values less than 4.6 (Figures 3, 4, and 5). Previous studies have described acidic pi values of 5.4 for HSP26 and pi ranging from 5.2 to 6.95 for HSP70 and HSP90 in HS quail red blood cells (Morimoto and Fodor, 1984; Dean and Atkinson, 1985). The present study, therefore, suggests that avian macrophages are sensitive to changes in their microenvironment both in vivo and in vitro. They respond by producing HS or stress proteins. Furthermore, as previously report-
ed, macrophages under HS conditions exhibit suppressed phagocytic potential (Miller and Qureshi, 1992a) as well as reduced synthesis of tumoricidal factor (Miller and Qureshi, 1992c). However, this suppression seems to be transient, because the phagocytic potential of macrophages returns to control levels after heat stress is removed. It is therefore speculated that the HS response may act as a physiological mechanism that protects macrophages from thermal and nonthermal damage and allows them to maintain their role in the immune functions under the most hostile conditions. ACKNOWLEDGMENTS The authors thank R. Hunter for excellent technical assistance and M. Robinson
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4*
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Kelley, P. M., and M. J. Schlesinger, 1978. The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell 15:1277-1286. Roller, M., and W. Konig, 1990. Arachidonic acid metabolism in heat-shock treated leucocytes. Immunology 70:458-464. Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriREFERENCES ophage T4. Nature 227:680-685. Banjeri, S. S., L. Berg, and R. I. Morimoto, 1986. Levinson, W., H. Opperman, and J. Jackson, 1978. Transcription and post-transcriptional regulaInduction of four proteins in eukaryotic cells by tion of avian HSP70 gene expression. J. Biol. kethoxal bis (thiosemicarbazone). Biochem. BioChem. 261:15740-15745. phys. Acta 518:401-412. Brush, A. H., 1965. Energetics, temperature regula- Levinson, W., H. Opperman, and J. Jackson, 1980. Transition series metals and sulfhydryl reagents tion and circulation in resting, active and induce the synthesis of four proteins in eudefeathered California quail, Lophortyx californikaryotic cells. Biochem. Biophys. Acta 606: cus. Comp. Biochem. Physiol. 15:399-421. 170-180. Ciavarra, R. P., and A. Simeone, 1990. T lymphocyte stress response. I. Induction of heat shock Lindquist, S., 1986. The heat-shock response. Annu. Rev. Biochem. 55:1151-1191. protein synthesis, a febrile temperature is correlated with enhanced resistance to hyperthermic Lindquist, S., and E. A. Craig, 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677. stress but not to heavy metal toxicity or dexamethasone-induced immune suppression. Mariodonneau-Parini, I., and B. S. Polla, 1987. Heat shock inhibits NADPH oxidase activity in Cell. Immunol. 129:363-376. human polymorphonuclear leukocytes. J. Cell. Clerget, M., and B. S. Polla, 1990. ErythrophagocytoBiol. 105:162a.(Abstr.) sis induces heat shock protein synthesis by human monocyte macrophages. Proc. Natl. Miller, L., and M. A. Qureshi, 1992a. Induction of heat-shock proteins and phagocytic function of Acad. Sci. USA 87:1081-1085. chicken macrophage following in vitro heat Dean, R. L., and B. G. Atkinson, 1985. Synthesis of exposure. Vet. Immunol. Immunopathol. 30: heat shock proteins in quail red blood cells 179-191. following brief, physiologically relevant increases in whole body temperature. Comp. Miller, L., and M. A. Qureshi, 1992b. Molecular changes associated with heat-shock treatment in Biochem. Physiol. 81:185-191. avian mononuclear and lymphoid lineage cells. Eid, N. S., R. E. Kravath, and K. W. Lanks, 1987. Poultry Sci. 71:473-481. Heat-shock protein synthesis by human polymorphonuclear cells. J. Exp. Med. 165: Miller, L., and M. A. Qureshi, 1992c. Comparison of heat-shock-induced and lipopolysaccharide1448-1452. induced protein changes and tumoricidal activFerris, D. K., A. Harel-Bellan, R. I. Morimoto, W. J. ity in chicken mononuclear cell line. Poultry Sci. Welch, and W. L. Farrar, 1988. Mitogen and 71:979-987. lymphokine stimulation of heat shock proteins in T lymphocytes. Proc. Natl. Acad. Sci. USA 85: Morimoto, R. I., and E. Fodor, 1984. Cell-specific expression of heat-shock proteins in chicken 3850-3854. reticulocytes and lymphocytes. J. Cell. Biol. 99: Hemmingsen, S., M. Woolford, S. M. Vandervies, K. 1316-1323. Tilly, D. T. Dennis, C. P. Georgopolus, R. W. Hendrix, and R. J. Ellis, 1988. Homologous plant National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy and bacterial proteins chaperone oligomeric Press, Washington, DC. protein assembly. Nature 333:330-334. CFarrell, P. H., 1975. High resolution twoHillman, P. E., N. R. Scott, and A. van Tienhoven, dimensional electrophoresis of proteins. J. Biol. 1985. Physiological responses and adaptations Chem. 250:4007-4021. to hot and cold environments. Pages 2-71 in: Phillips, H. J., 1973. Dye exclusion test for cell Stress Physiology in Livestock Volume HI, viability. Page 406 in: Culture Methods and Poultry. M. K. Yousef, ed. CRC Press Inc., Boca Applications. P. F. Kruse, Jr. and M. K. PatterRaton, FL. son, Jr., ed. Academic Press, New York, NY. Hudson, J. W., and A. H. Brush, 1964. A comparative Polla, B. S., J. V. Bonventre, and S. M. Krane, 1988. study of cardiac and metabolic performance of 1,25-dihydroxyvitamin D3 increases the toxicity the dove Zenaidura macroura, and the quail, of hydrogen peroxide in the human monocytic Lophortyx californicus. Comp. Biochem. Physiol. line U937: The role of calcium and heat shock. J. 12:157-170. Cell Biol. 107:373-380. Johnston, D., H. Opperman, J. Jackson, and W. Polla, B. S., A. M. Healy, W. C. Wojno, and S. M. 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and C. Smith for typing the manuscript. The research reported in this publication was funded by North Carolina Agricultural Research Services, Project Number NC06175.
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