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Macrophage culture: influence of species-specific incubation temperature
Journal of Immunological Methods 214 Ž1998. 165–174
Macrophage culture: influence of species-specific incubation temperature Valerie A.I. Natale, Kenneth C. McCullough
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Institute of Virology and Immunoprophylaxis, Postfach, CH-3147, Mittelhausern, Switzerland ¨ Received 20 November 1997; revised 18 February 1998; accepted 5 March 1998
Abstract Cultured mammalian cells are traditionally maintained at 378C, despite the fact that core body temperatures differ considerably among mammals. Considering the body temperature of the adult pig, comparison was made of porcine macrophage cultures maintained at 378C and 39.28C. Examination of the cells showed that granularity was higher in macrophages maintained at 39.28C, although no differences in cell size were observed. The density of MHC Class I and II expression was higher on cells maintained at 39.28C, as was the percentage of MHC Class II positive cells. In contrast, expression of CD44 and CD11ar18 remained unchanged. Following stimulation with lipopolysaccharide, only cells maintained at 39.28C produced detectable levels of TNF-a . As a final reference criterion, replication of the macrophage tropic African swine fever virus was monitored. At 39.28C, virus antigen production was less efficient, and virus isolate-related differences in the replication kinetics were observed. Infectious virus production was not different at the two temperatures, implying that virus maturation may have been more efficient at the higher temperature. These results indicate that incubation of cultured cells at the temperature of their donor species has an important influence on their characteristics. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Porcine monocytic cells; Temperature; African swine fever virus
1. Introduction Animal cell culture is a widely used technique in all areas of biological research. Cultured mammalian cells are traditionally maintained in incubators set at 378C, in spite of the fact that body temperatures of non-human mammals are generally 1 to 2.58C higher than 378C. In fact, a review of the extensive number of studies that have researched or used cultured
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mammalian cells, shows that incubators are set to 378C as a matter of course during culture of cells derived from cows ŽRey Nores et al., 1995; Stone et al., 1995; Yang et al., 1996., dogs ŽNakada et al., 1996., rabbits ŽMacen et al., 1996., sheep ŽCoughlin et al., 1996; Wunderlin and Palmer, 1996., pigs ŽEnjuanes et al., 1977; Malmquist and Hay, 1960; Plowright and Parker, 1967., and even deer ŽCross et al., 1996.. Indeed, textbooks and manuals dedicated to the subject of animal cell culture instruct readers to set incubator temperatures to 378C ŽGriffiths, 1992; Morgan and Darling, 1993.. Although some authors of studies utilising mammalian cells have incubated
0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 5 5 - 6
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cells at temperatures higher than 378C ŽRivas et al., 1995., the incubator temperatures used in the studies were nonetheless lower than temperatures given for the donor animals ŽFraser, 1991.. Furthermore, there do not appear any studies comparing the effects of species-specific incubator temperature with incubations at 378C. Some investigators do not even report incubator temperature ŽEven et al., 1995; Land et al., 1996; Magyar et al., 1995; Meinkoth and Meyers, 1995; Mirsky et al., 1990; Yang et al., 1996.. This omission implies that temperature is not perceived as an important factor in animal cell culture. African swine fever, or ASF, is a haemorrhagic disease caused by a large DNA virus that is, to date, the only known member of the group African swine fever-like viruses ŽDixon et al., 1995; Montgomery, 1921.. The virus’ host range is restricted to members of the Suidæ family, and ticks of the family Ornithodoros ŽHess et al., 1971; Montgomery, 1921; Sanchez-Botija, 1963; Wardley and Wilkinson, 1977.. In swine, the virus infects mononuclear phagocytes, fibroblasts, smooth muscle cells, interstitial endothelial cells, and secondary lymphoid organs ŽCasal et al., 1984; Fernandez et al., 1992; GomezVillamandos et al., 1995; Mebus, 1988; Vinuela, ˜ 1987.. Due to the lack of model systems with the virus, many studies of ASF and ASF virus are performed in vitro, using monocytic cells taken from donor animals. Variations in the susceptibility to infection of monocytic cells have been reported, and have been related to the culturing of the monocytes, the anatomical source of the cells, or the presence of colony stimulating factor-like activity in the medium ŽGenovesi et al., 1990; Malmquist and Hay, 1960; McCullough et al., 1993.. The purpose of this study was to investigate the influence of incubator temperature upon cultured porcine leukocytes, with a view to establishing the temperature for optimal incubation of such cells, and perhaps indicating how other mammalian cells should be handled in vitro. Thus, comparative assays of a number of parameters were made, including virus infection, cell granularity, cell phenotype, and production of TNF-a . Data obtained from these studies were used to support the hypothesis that culture of porcine leukocytes, and possibly other mammalian cells at 378C results in sub-optimal culture conditions for the cells.
2. Materials and methods 2.1. Media, buffers and supplements High glucose DMEM Ž5000 mgrml, Gibco. and pyrogen-free water were employed. Phenol red-free medium was used in order to avoid phagocytosis of phenol red by macrophages, and subsequent undesired fluorescence during immunofluorescence analyses. Porcine plasma from heparinized blood was added at 30% vrv, to give the ‘growth medium’ necessary for the monocyte cultures in which cells would differentiate into macrophages. Without this porcine plasma supplement, porcine monocytes will not differentiate into macrophages in vitro; for example, foetal calf serum is unsuitable. PBS ŽDulbecco’s m o d ific a tio n ., P B S r A -E D T A Žcalciumrmagnesium-free PBS containing 0.03% wrv EDTA. and Alsever’s solution were prepared from the basic chemical ingredients using sterile pyrogen-free water. All glassware was sterilised at 1808C for 6 h. Sterile, disposable plastic pipettes and pipette tips were used throughout the course of experimentation. Plastic culture flasks and plates were obtained from Costar ŽCambridge, MA, USA.. 2.2. Cells Citrated vena caval blood samples were obtained weekly from two of four blood donor Swiss Landrace pigs. Mononuclear cells were isolated using a modification of Bøyum’s method ŽBøyum, 1960.. Whole blood samples were centrifuged at 1000 = g for 30 min to obtain buffy coats, which were then centrifuged at 800 = g for 20 min through a Ficoll– Hypaque gradient. Peripheral blood mononuclear cells ŽPBMC. were washed by centrifugation in PBSrA-EDTA at 350 = g for 20 min at room temperature. They were then centrifuged twice at 250 = g for 10 min at 48C to remove platelets. PBMCs were resuspended at a concentration of 4 = 10 6 cells per milliliter in DMEM containing 10% Žvrv. FCS ŽSebak.. Monocytic cells were adhered to the surface of the plastic tissue culture vessel by incubation at 378C, and separated from non-adherent lymphocytic cells by washing with pre-warmed PBSr1% Žvrv. FCS. ŽSimilar results were obtained if adherence was
V.A.I. Natale, K.C. McCulloughr Journal of Immunological Methods 214 (1998) 165–174
at 39.28C.. The adherent cells were dominated by monocytes, characterized by labelling with antibody against the pan-myeloid marker for porcine cells SWC3, and the SWC1 marker found on monocytes and T lymphocytes ŽLunney, 1993; Saalmuller, 1996; ¨ McCullough et al., 1997.. Growth medium was added to the adherent cell cultures, which were then incubated at either 378C or 39.28C in a humidified atmosphere with 6% Žvrv. CO 2 until ready for use. Cells were examined at three to four days post-plating, at which time they had differentiated into monocyte-derived macrophages, regardless of their temperature of incubation. Greater than 95% of the adherent cells were macrophages, defined by the recently identified SWC9 mature porcine macrophage marker ŽSaalmuller, 1996; McCullough et al., 1997.. ¨ 2.3. Phenotyping Phenotype assays were performed on live cells using the FACScan flow cytometer, at the time points indicated in Section 2.4. Cells were removed from culture flasks by scraping into cold PBS containing 20-mM sodium azide. Propidium iodide was used to exclude dead cells. Monoclonal antibodies directed against CD44, CD11ar18, MHC class I, MHC class II, and the SWC3 porcine myeloid marker were used ŽLunney, 1993.. The myeloid marker permitted the exclusion of any contaminating nonmyeloid cells during analysis. SWC1 was also employed to identify SWC3q monocytes Žthis SWC1 is not expressed on mature macrophages; McCullough et al., 1997.. For macrophage differentiation, the SWC9 marker, found on mature macrophages but not monocytes, was employed ŽMcCullough et al., 1997.. Antibodies were diluted 1r50, and incubated with cells for 30 min at 48C; the cells were then washed for 8 min at 350 = g with a 1-min brake. FITC-conjugated rabbit FŽabX . 2 anti-mouse immunoglobulins ŽDako, Zug, Switzerland. were added next, diluted at 1r100, for 30 min at 48C. Cells were re-washed, and diluted in PBS containing 20-mM sodium azide. Propidium iodide was added to the cells Žafter dilution of a 100 m grml stock solution to 1r100. immediately prior to FACS analysis. SWC3-labelled cells were selected and gated in a fluorescence dot-plot of cells. This selection of cells was subsequently coloured in a size-granularity plot
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of the same cells. A gate was drawn around these cells, and this gated population defined myeloid cells in subsequent fluorescence histograms. Two parameters were employed during the analysis of phenotype, namely, the percentage of positive cells Žrelative to the negative peak. and the intensity values. Intensity values describe the relative number of bound antibodies per cell, relative to the negative peak. Analysis employed a Becton Dickinson FACScan flow cytometer and Lysis II software. 2.4. ActiÕation of monocytic cells with lipopolysaccharide (LPS) and quantification of TNF-a Cells were stimulated with 1 m grml of lipopolysaccharide ŽLPS. at 3 days post-plating. Supernatant samples were taken from cell cultures 24 h later, and were frozen at y708C until analysis. TNF in the supernatants was quantified using a porcine TNF ELISA kit ŽEndogen, Cambridge, MA.. Briefly, samples were added to wells that were pre-coated with anti-TNF antibody, in a 96-well plate. Biotinylated anti-TNF antibody was added 30 min later, and the plate was incubated for 2 h at room temperature. The plate was rinsed and streptavidin was added for 30 min. Enzymatic colour reagent was added and plates were developed in the dark. Stop solution was added, and the plate was read immediately with an ELISA reader set to 450 and 550 nm. Values from 550 nm were subtracted from values obtained at 450 nm. Values of controls were plotted on a standard curve and sample values were read from the curve. 2.5. Virus Four isolates of ASF virus were used for this study; these isolates were kindly provided by Dr. P. Wilkinson, IAH, Pirbright, UK. They were KWH Žmoderately virulent., Malawi Žhighly virulent., Perpignan Žalso highly virulent., and CV-1 Žavirulent in pigs.. Each of the virulent isolates had been passaged at least once in pigs, to provide a spleen or lymph node extract of ‘in vivo stock’ virus. The Perpignan virus was also available as an infectious blood preparation: lysed whole blood collected from an animal with acute clinical symptoms of ASF. Additional ‘in vitro stocks’ of these viruses were prepared by passaging them twice in cultured swine macrophages
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Žincubation temperature 378C., at input multiplicities of 0.001 immunofluorescence focus-forming units ŽIFFU. per cell. The CV-1 strain had been adapted to and passaged in CV-1 cells, and finally in swine macrophages Žincubation temperatures 378C.. Titres were expressed in IFFU per milliliter. The multiplicity of infection Žm.o.i.. for experiments was 0.5–1.0 IFFU per cell.
tein VP32, and an FITC-labelled isotype-specific conjugate. Anti-VP32 was diluted 1r1000, and was the gift of Dr. F. Alonso, CISA-INIA, Madrid, Spain. The conjugate was diluted 1r50 and was obtained from Southern Biotechnology, USA. Analysis employed a Becton Dickinson FACScan flow cytometer and Lysis II software.
2.6. Detection of Õirus infection
3. Results
Infected or uninfected control cells were removed from culture flasks by scraping at 24 h post-infection, and were fixed. Fixation was performed using a cell fixation and permeabilisation kit according to the manufacturer’s instructions ŽHarlan Sera-Lab, Sussex, UK.. They were labelled for flow cytometry using a monoclonal antibody against ASF virus pro-
3.1. Effect of temperature on size and granularity of cells Statistics from flow cytometer forward- and sidescatter plots were used to compare size and granularity of cells maintained at the two temperatures. No consistent differences in cell size were observed over
Fig. 1. Comparison of the size Žforward scatter, FSC, x-axis. and granularity Žside scatter, SSC, y-axis. of porcine monocyte-derived macrophages maintained at 378C Ža, c. or 39.28C Žb, d., either untreated Ža, b. or stimulated for 24 h with 1 m grml LPS Žc, d..
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Fig. 2. Phenotype of the monocytes isolated from porcine peripheral blood and used for the generation of macrophages in the experiments shown in the other figures. Ža. Percentage of cells Ž y-axis. clearly positive for the cell surface marker Žshown on the x-axis.. Žb. Relative intensity of fluorescence staining Ž y-axis. for each of the markers Žshown on the x-axis.. The values represent the mean from three different analyses Ždifferent preparations of blood monocytes. " SEM.
the course of various experiments. However, macrophages that had been incubated at 39.28C were more granular than cells maintained at 378C. Fig. 1a and b shows that granularity in unstimulated macrophages was higher in cells kept at 39.28C. Similarly, granularity was higher in cells incubated at the higher temperature and stimulated with 1 m grml of LPS ŽFig. 1c and d.. LPS-stimulated cells were examined 24 h post-stimulation. These experiments were performed three times; unstimulated cells kept at 398C were approximately 12% more granular than cells kept at 378C, and LPS-stimulated cells
kept at 398C were approximately 9% more granular than stimulated cells kept at 378C. 3.2. Effect of temperature on cell phenotype Cells were isolated as described in Section 2, plated in 75 cm2 plastic culture flasks, and incubated for 3 days at 378C or 39.28C, to allow differentiation into macrophages. They were removed from culture flasks at 3 days post-plating by scraping in PBS containing sodium azide, and labelled with monoclonal antibodies against MHC classes I and II,
Fig. 3. Expression of MHC class II on porcine monocyte-derived macrophages incubated at 378C or 39.28C. Ža. Percentage of cells Ž y-axis. recorded as MHC Class II positive; Žb. the relative fluorescence intensity Ž y-axis. of MHC class II expression on the positive cells. The x-axis shows the experiment number which generated the results.
V.A.I. Natale, K.C. McCulloughr Journal of Immunological Methods 214 (1998) 165–174
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Table 1 Fluorescence intensity per cell a of the expression of MHC Class I, CD11ar18 and CD44 on porcine monocyte-derived macrophages maintained at 378C and 39.28C Cell surface antigen MHC Class I CD11ar18 CD44
Experiment 1 378C 39.28C
Experiment 2 378C 39.28C
Experiment 3 378C 39.28C
117 a 39 581
136 48 479
154 40 209
142 34 593
174 51 505
196 40 282
a Measurements were "10% coefficient of variation within experiments; for reference, the negative control labellings gave values -10.
CD11ar18, CD18, CD44 and the SWC3 myeloid marker. For comparison, the monocytes employed for the generation of these macrophages were similarly phenotyped ŽFig. 2.. These experiments showed that the expression of certain surface molecules was higher in cells incubated at 39.28C than in cells incubated at 378C. For example, Fig. 3a shows that a higher percentage of cells cultured at 39.28C expressed MHC class II, although the increase was slight in experiment number 5. Furthermore, the intensity of fluorescence per cell was generally higher in the cells maintained at 39.28C ŽFig. 3b.. Similarly, MHC class I fluorescence intensity was up-regulated in cells incubated at 39.28C. A summary of the results from three experiments are shown in Table 1. However, the 39.28C cells in experiment number 5 Žsee Fig. 3b. did not show a significant increase over the 378C cells. Values for the percentage of cells expressing the antigens shown in Table 1 are not shown because nearly 100% of cells kept at each temperature expressed these antigens. Table 1 also shows data for CD11ar18 and CD44 antigens. Fluorescence intensity values for these molecules did not significantly differ between cultures maintained at the two temperatures. Intensity values between cells maintained at the two temperatures and labelled with CD18 were variable Žresults not shown.. 3.3. Influence of temperature on production of tumour necrosis factor A porcine TNF ELISA was used as described in Section 2 to determine if temperature affected pro-
Table 2 Production of TNF-a is greater by macrophages maintained at 39.28C than at 378C Experiment
1 2 3 4
TNF-a Žpgrml. 378C
39.28C
0 0 51 41
106 145 118 51
The TNF was quantified in pgrml, and measured using a commercial ELISA kit for porcine TNF.
duction of TNF-a in stimulated macrophages. Cells isolated from the same animal were incubated at 378C or 39.28C. On the third day in vitro, they were stimulated with 1 m grml of LPS. Samples of the culture medium were removed at 24 h post-stimulation and stored at y708C until analysis. Table 2 shows levels of TNF-a produced in the cells during four experiments. TNF-a levels were very low or undetected in media from all cells maintained at 378C. However, the cytokine was detected in all of four media samples taken from cells incubated at the higher temperature. Thus, incubation at the lower temperature does not facilitate efficient TNF-a production by LPS-stimulated macrophages. 3.4. Effect of temperature on ASF Õirus replication Porcine mononuclear cells, isolated and incubated as described in the Section 2, were plated in 25 cm2 plastic culture flasks. At 3 days post-plating, they were infected simultaneously with the KWH isolate of ASF virus, at an m.o.i. of 0.5–1.0 IFFU per cell. Infected cells were detected flow cytometrically at 24 h post-infection, using the monoclonal antibody anti-ASFV VP32, as described in the Section 2. Cells from flasks maintained at different temperatures were fixed simultaneously. Fig. 4a shows data from four experiments. In each case, the percentage of antigen-positive cells in the 378C-cultures was higher than that in 39.28C-cultures. Furthermore, the difference in the proportion of infected cells between the two temperatures was nearly 40% in three out of four experiments. No differences in extracellular virus titre were observed between cultures maintained at the two temperatures Ždata not shown..
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Fig. 4. Replication of ASF virus in porcine monocyte-derived macrophages prepared and maintained at 378C or 39.28C. Cells were infected with 0.5 to 1.0 IFFUrcell, and analysed at 24 h post-infection for the presence of ASF virus-specific antigen ŽVP32. production Ž y-axis; ‘percentage infected cells’.. Ža. Infection with the moderately virulent KWH isolate of ASF virus; Žb. infections with the virulent Malawi ŽM. isolate, virulent Perpignan ŽP. isolate or cell-culture adapted avirulent CV-1 ŽCV. strain. The x-axis shows the number of the experiment which generated the results, and the virus used Žin panel b..
Infection detection experiments were repeated using three other isolates of ASF virus, in order to determine if in vivo virulence of the infecting virus would influence the outcome of the experiment. These isolates were Malawi, Perpignan, and CV-1. The m.o.i. and method for virus detection were identical to those used for the KWH isolate experiments. Each experiment with a separate virus was performed twice. As shown in Fig. 4b, more cells in the 378C cultures were antigen-positive at 24 h postinfection, regardless of the virus infecting them. Both the Malawi and Perpignan isolates were less efficient at producing antigen than CV-1 strain at 39.28C, compared to 378C. Such results could not be explained by the virus having been propagated in macrophage cultures at 378C. Both the in vitro propagated and the ‘in vivo’ stock of Perpignan virus— the latter prepared as lysed blood from an acutely infected pig—gave similar results to those in Fig. 4b. This finding implies that viruses may show differences in their abilities to replicate that are not evident when cells are maintained at 378C.
4. Discussion The body temperatures of most mammals are higher than 378C, and those of most birds, including chickens, are over 408C. Hypothermia in a human is
described as a drop in body temperature of 28C ŽTortora and Grabowski, 1993.. Thus, incubation of cells such as porcine cells, at 378C can result in the establishment of a mild hypothermic state. A wellknown characteristic of this condition is a decrease in cellular metabolism—including the rates of biochemical reactions—and a reduction in oxygen availability in tissues that results from the disruption of normal haemoglobin–oxygen dissociation ŽTortora and Grabowski, 1993; Radostits et al., 1994; Schubert, 1995.. The reduction in metabolic rate is approximately 5% to 7% per degree Celsius ŽReuler, 1978; Schubert, 1995.. All of these reductions would have a negative effect upon cellular function. The data presented in this paper show that a number of cell functions were reduced at 378C, notably the expression of MHC but not adhesion molecules, the production of TNF-a , and macrophage-tropic virus replication. The increased percentage of ASF virus antigenpositive cells at 378C may have been due to a number of factors. For instance, the increased granularity in the 39.28C cells implies that organelles such as lysosomes andror secretory vesicles may have increased in number or activity. A greater number of these organelles would mean an increase in antimicrobial enzymes available to the cells. Variations between the virus isolates in their optimal temperatures for replication have been observed
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with a number of viruses ŽAynaud et al., 1972; Hozinsky et al., 1966; Lwoff and Lwoff, 1960; MacKenzie, 1969.. For example, virulent isolates of classical swine fever virus replicated to highest titre in PK-15 cells maintained at 39.28C to 408C, while avirulent isolates preferred an environment of 338C to 348C ŽAynaud et al., 1972.. Isolates of low virulence replicated optimally at temperatures between those for the virulent and avirulent isolates ŽAynaud et al., 1972.. The authors of the study noted that the optimal replication temperature of the virulent isolates corresponded to the body temperature of a domestic pig. These observations imply that ASF virus might also replicate preferentially at higher temperature. However, the present study showed that ASF virus VP32 antigen production was detected in fewer cells from the 39.28C cultures at 24 h post-infection, but there was no difference in extracellular virus titre obtained from cultures maintained at the two temperatures. These observations are in contrast to those for classical swine fever virus. They would imply that antigen production is more effective at the lower temperature but virus maturation and assembly could be most efficient at the body temperature of the pig. An explanation for these apparently contradictory results may lie in the type of cells used to propagate the viruses. The virus isolates used in the classical swine fever study were all cultivated in PK-15 cell lines that had been passaged repeatedly at 378C. Such multiple passaging of the cells may have caused them to adapt to the lower temperature, and certain critical functional capabilities at the higher temperatures might have been impaired, allowing the virulent isolate to replicate to a higher titre. Alternatively, the increase in temperature to 39.28C may have allowed certain functions to increase in the cells. Furthermore, PK-15 cells are not intrinsically destructive, as are macrophages. Finally, the virulent classical swine fever virus isolates may, as the authors suggested, have been better suited to replicate at the temperature corresponding to the susceptible animal, at least in terms of infectious progeny production. ASF virus may also be better suited to replicate at the temperature of the susceptible animal. The data presented in this study showed that primary cultures of porcine macrophages function differently at 39.28C compared to 378C: they were
more granular, and they produced more TNF-a upon stimulation. These factors may explain why less ASF virus antigen was detected at 24 h post-infection: macrophages specifically destroy invading pathogens, and the culture of porcine macrophages at a temperature that is more than 28C lower than that to which they have adapted may well impair some of their functionality. This fact is of crucial importance to the study of the biological and biochemical characteristics of cultured cells: the use and study of cells maintained at a sub-optimal temperature will, in all likelihood, result in the generation of misleading results. These results may be related to pathogen infection, cytokine production, enzyme function, drug function, protein synthesis, production of new cell lines, or any number of parameters. The production of TNF-a by the cells used in this study illustrates this point elegantly. Cells maintained at 378C produced little or no TNF-a , in sharp contrast to cells incubated at 39.28C. Thus, incubation of cultured cells at the temperature of their donor species is a vital factor in their culture, and analytical applications.
Acknowledgements This work was supported by a Swiss Nationalfonds grant, number NF 31-33646.92. We wish to thank Rene´ Schaffner for technical assistance, and Dr. Christian Griot for interesting discussions.
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Malmquist, W.A., Hay, D., 1960. Hæmadsorption and cytopathic effect produced by African swine fever virus in swine bone marrow and buffy coat cultures. Am. J. Vet. Res. 21, 104–108. McCullough, K.C., Schaffner, R., Fraefel, W., Kihm, U., 1993. The relative density of CD44-positive porcine monocytic cell populations varies between isolations and upon culture, and influences susceptibility to infection by African swine fever virus. Immunol. Lett. 37, 83–90. McCullough, K.C., Schaffner, R., Kihm, Y., Natale, V.A.I., Summerfield, A., 1997. The phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages. Vet. Immunol. Immunopathol. 58, 265–275. Mebus, C.A., 1988. African swine fever. Adv. Virus Res. 35, 251–269. Meinkoth, J.H., Meyers, K.M., 1995. Measurement of von Willebrand factor-specific mRNA and release and storage of von Willebrand factor from endothelial cells of dogs with type-I von Willebrand’s disease. Am. J. Vet. Res. 56, 1577–1585. Mirsky, M.L., Olmstead, C.A., Da, Y., Lewin, H.A., 1990. The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection. J. Virol. 70, 2178–2183. Montgomery, R.E., 1921. On a form of swine fever occurring in British East Africa ŽKenya Colony.. J. Comp. Pathol. Ther. 34 Ž159–191., 243–262. Morgan, S.J., Darling, D.C., 1993. Animal Cell Culture. ßios scientific publishers. Nakada, Y., Soga, M., Kosaka, T., Tsukatani, Y., Miyamori, M., Kuwabara, M., Tanaka, S., Koide, F., Fujiwara, K., 1996. Characterization of natural killer cytotoxic factor ŽNKCF. from canine NK cells. Vet. Immunol. Immunopathol. 49, 283–293. Plowright, W., Parker, J., 1967. The stability of African swine fever virus with particular reference to heat and pH inactivation. Arch. Gesamte Virusforsch 21, 383–402. Radostits, O.M., Blood, D.C., Gay, C.C. 1994. Veterinary Medicine, 8th edn. Balliere ` Tindall, London. Reuler, J.B., 1978. Hypothermia: pathophysiology, clinical settings, and management. Ann. Intern. Med. 89, 519–527. Rey Nores, J., Anderson, J., Butcher, R., Libeau, G., McCullough, K.C., 1995. Rinderpest virus infection of bovine peripheral blood monocytes. J. Gen. Virol. 76, 2779–2791. Rivas, A.L., Kimball, E.S., Quimby, F.W., Gebhard, D., 1995. Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 45, 55–71. Saalmuller, A., 1996. Characterization of swine leukocyte differ¨ entiation antigens. Immunol. Today 17, 352–354. Sanchez-Botija, C., 1963. Reservorios del virus de la peste porcina Africana: investigacion del virus de la P.P.A. en los artropodos mediante la prueba de la hemadsorcion. Bull. Off. Int. Epizoot. 60, 895–899. Schubert, A., 1995. Side effects of mild hypothermia. J. Neurosurg. Anesthesiol. 7, 139–147. Stone, D.M., Hof, A.J., Davis, W.C., 1995. Up-regulation of IL2 receptor a and MHC class II expression on lymphocyte sub-
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populations from bovine leukemia virus infected lymphocytic cows. Vet. Immunol. Immunopathol. 48, 65–76. Tortora, G.J., Grabowski, S.R., 1993. Principles of Anatomy and Physiology, 7th edn. HarperCollins College Publishers, USA. Vinuela, E., 1987. Molecular biology of African swine fever ˜ virus. In: Becker, Y. ŽEd.., African Swine Fever Virus. Martinus Nijhoff, Boston, pp. 31–50. Wardley, R.C., Wilkinson, P.J., 1977. The growth of virulent African swine fever virus in pig monocytes and macrophages. J. Gen. Virol. 38, 183–186.
Wunderlin, E., Palmer, D.G., 1996. Measurement of an eosinophil differentiation factor in sheep before and after infection with Trichostrongylus colubriformis. Vet. Immunol. Immunopathol. 49, 169–175. Yang, Z., Breider, M.A., Carroll, R.C., Miller, M.S., Bochsler, P.N., 1996. Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharidemediated cytotoxicity and activation of bovine vascular endothelial cells in vitro. J. Leukocyte Biol. 59, 241–247.
Macrophage culture: influence of species-specific incubation temperature Valerie A.I. Natale, Kenneth C. McCullough
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Institute of Virology and Immunoprophylaxis, Postfach, CH-3147, Mittelhausern, Switzerland ¨ Received 20 November 1997; revised 18 February 1998; accepted 5 March 1998
Abstract Cultured mammalian cells are traditionally maintained at 378C, despite the fact that core body temperatures differ considerably among mammals. Considering the body temperature of the adult pig, comparison was made of porcine macrophage cultures maintained at 378C and 39.28C. Examination of the cells showed that granularity was higher in macrophages maintained at 39.28C, although no differences in cell size were observed. The density of MHC Class I and II expression was higher on cells maintained at 39.28C, as was the percentage of MHC Class II positive cells. In contrast, expression of CD44 and CD11ar18 remained unchanged. Following stimulation with lipopolysaccharide, only cells maintained at 39.28C produced detectable levels of TNF-a . As a final reference criterion, replication of the macrophage tropic African swine fever virus was monitored. At 39.28C, virus antigen production was less efficient, and virus isolate-related differences in the replication kinetics were observed. Infectious virus production was not different at the two temperatures, implying that virus maturation may have been more efficient at the higher temperature. These results indicate that incubation of cultured cells at the temperature of their donor species has an important influence on their characteristics. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Porcine monocytic cells; Temperature; African swine fever virus
1. Introduction Animal cell culture is a widely used technique in all areas of biological research. Cultured mammalian cells are traditionally maintained in incubators set at 378C, in spite of the fact that body temperatures of non-human mammals are generally 1 to 2.58C higher than 378C. In fact, a review of the extensive number of studies that have researched or used cultured
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Corresponding author. Tel.: q41-31-848-9361; fax: q41-31848-9222; e-mail: [email protected].
mammalian cells, shows that incubators are set to 378C as a matter of course during culture of cells derived from cows ŽRey Nores et al., 1995; Stone et al., 1995; Yang et al., 1996., dogs ŽNakada et al., 1996., rabbits ŽMacen et al., 1996., sheep ŽCoughlin et al., 1996; Wunderlin and Palmer, 1996., pigs ŽEnjuanes et al., 1977; Malmquist and Hay, 1960; Plowright and Parker, 1967., and even deer ŽCross et al., 1996.. Indeed, textbooks and manuals dedicated to the subject of animal cell culture instruct readers to set incubator temperatures to 378C ŽGriffiths, 1992; Morgan and Darling, 1993.. Although some authors of studies utilising mammalian cells have incubated
0022-1759r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 0 5 5 - 6
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cells at temperatures higher than 378C ŽRivas et al., 1995., the incubator temperatures used in the studies were nonetheless lower than temperatures given for the donor animals ŽFraser, 1991.. Furthermore, there do not appear any studies comparing the effects of species-specific incubator temperature with incubations at 378C. Some investigators do not even report incubator temperature ŽEven et al., 1995; Land et al., 1996; Magyar et al., 1995; Meinkoth and Meyers, 1995; Mirsky et al., 1990; Yang et al., 1996.. This omission implies that temperature is not perceived as an important factor in animal cell culture. African swine fever, or ASF, is a haemorrhagic disease caused by a large DNA virus that is, to date, the only known member of the group African swine fever-like viruses ŽDixon et al., 1995; Montgomery, 1921.. The virus’ host range is restricted to members of the Suidæ family, and ticks of the family Ornithodoros ŽHess et al., 1971; Montgomery, 1921; Sanchez-Botija, 1963; Wardley and Wilkinson, 1977.. In swine, the virus infects mononuclear phagocytes, fibroblasts, smooth muscle cells, interstitial endothelial cells, and secondary lymphoid organs ŽCasal et al., 1984; Fernandez et al., 1992; GomezVillamandos et al., 1995; Mebus, 1988; Vinuela, ˜ 1987.. Due to the lack of model systems with the virus, many studies of ASF and ASF virus are performed in vitro, using monocytic cells taken from donor animals. Variations in the susceptibility to infection of monocytic cells have been reported, and have been related to the culturing of the monocytes, the anatomical source of the cells, or the presence of colony stimulating factor-like activity in the medium ŽGenovesi et al., 1990; Malmquist and Hay, 1960; McCullough et al., 1993.. The purpose of this study was to investigate the influence of incubator temperature upon cultured porcine leukocytes, with a view to establishing the temperature for optimal incubation of such cells, and perhaps indicating how other mammalian cells should be handled in vitro. Thus, comparative assays of a number of parameters were made, including virus infection, cell granularity, cell phenotype, and production of TNF-a . Data obtained from these studies were used to support the hypothesis that culture of porcine leukocytes, and possibly other mammalian cells at 378C results in sub-optimal culture conditions for the cells.
2. Materials and methods 2.1. Media, buffers and supplements High glucose DMEM Ž5000 mgrml, Gibco. and pyrogen-free water were employed. Phenol red-free medium was used in order to avoid phagocytosis of phenol red by macrophages, and subsequent undesired fluorescence during immunofluorescence analyses. Porcine plasma from heparinized blood was added at 30% vrv, to give the ‘growth medium’ necessary for the monocyte cultures in which cells would differentiate into macrophages. Without this porcine plasma supplement, porcine monocytes will not differentiate into macrophages in vitro; for example, foetal calf serum is unsuitable. PBS ŽDulbecco’s m o d ific a tio n ., P B S r A -E D T A Žcalciumrmagnesium-free PBS containing 0.03% wrv EDTA. and Alsever’s solution were prepared from the basic chemical ingredients using sterile pyrogen-free water. All glassware was sterilised at 1808C for 6 h. Sterile, disposable plastic pipettes and pipette tips were used throughout the course of experimentation. Plastic culture flasks and plates were obtained from Costar ŽCambridge, MA, USA.. 2.2. Cells Citrated vena caval blood samples were obtained weekly from two of four blood donor Swiss Landrace pigs. Mononuclear cells were isolated using a modification of Bøyum’s method ŽBøyum, 1960.. Whole blood samples were centrifuged at 1000 = g for 30 min to obtain buffy coats, which were then centrifuged at 800 = g for 20 min through a Ficoll– Hypaque gradient. Peripheral blood mononuclear cells ŽPBMC. were washed by centrifugation in PBSrA-EDTA at 350 = g for 20 min at room temperature. They were then centrifuged twice at 250 = g for 10 min at 48C to remove platelets. PBMCs were resuspended at a concentration of 4 = 10 6 cells per milliliter in DMEM containing 10% Žvrv. FCS ŽSebak.. Monocytic cells were adhered to the surface of the plastic tissue culture vessel by incubation at 378C, and separated from non-adherent lymphocytic cells by washing with pre-warmed PBSr1% Žvrv. FCS. ŽSimilar results were obtained if adherence was
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at 39.28C.. The adherent cells were dominated by monocytes, characterized by labelling with antibody against the pan-myeloid marker for porcine cells SWC3, and the SWC1 marker found on monocytes and T lymphocytes ŽLunney, 1993; Saalmuller, 1996; ¨ McCullough et al., 1997.. Growth medium was added to the adherent cell cultures, which were then incubated at either 378C or 39.28C in a humidified atmosphere with 6% Žvrv. CO 2 until ready for use. Cells were examined at three to four days post-plating, at which time they had differentiated into monocyte-derived macrophages, regardless of their temperature of incubation. Greater than 95% of the adherent cells were macrophages, defined by the recently identified SWC9 mature porcine macrophage marker ŽSaalmuller, 1996; McCullough et al., 1997.. ¨ 2.3. Phenotyping Phenotype assays were performed on live cells using the FACScan flow cytometer, at the time points indicated in Section 2.4. Cells were removed from culture flasks by scraping into cold PBS containing 20-mM sodium azide. Propidium iodide was used to exclude dead cells. Monoclonal antibodies directed against CD44, CD11ar18, MHC class I, MHC class II, and the SWC3 porcine myeloid marker were used ŽLunney, 1993.. The myeloid marker permitted the exclusion of any contaminating nonmyeloid cells during analysis. SWC1 was also employed to identify SWC3q monocytes Žthis SWC1 is not expressed on mature macrophages; McCullough et al., 1997.. For macrophage differentiation, the SWC9 marker, found on mature macrophages but not monocytes, was employed ŽMcCullough et al., 1997.. Antibodies were diluted 1r50, and incubated with cells for 30 min at 48C; the cells were then washed for 8 min at 350 = g with a 1-min brake. FITC-conjugated rabbit FŽabX . 2 anti-mouse immunoglobulins ŽDako, Zug, Switzerland. were added next, diluted at 1r100, for 30 min at 48C. Cells were re-washed, and diluted in PBS containing 20-mM sodium azide. Propidium iodide was added to the cells Žafter dilution of a 100 m grml stock solution to 1r100. immediately prior to FACS analysis. SWC3-labelled cells were selected and gated in a fluorescence dot-plot of cells. This selection of cells was subsequently coloured in a size-granularity plot
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of the same cells. A gate was drawn around these cells, and this gated population defined myeloid cells in subsequent fluorescence histograms. Two parameters were employed during the analysis of phenotype, namely, the percentage of positive cells Žrelative to the negative peak. and the intensity values. Intensity values describe the relative number of bound antibodies per cell, relative to the negative peak. Analysis employed a Becton Dickinson FACScan flow cytometer and Lysis II software. 2.4. ActiÕation of monocytic cells with lipopolysaccharide (LPS) and quantification of TNF-a Cells were stimulated with 1 m grml of lipopolysaccharide ŽLPS. at 3 days post-plating. Supernatant samples were taken from cell cultures 24 h later, and were frozen at y708C until analysis. TNF in the supernatants was quantified using a porcine TNF ELISA kit ŽEndogen, Cambridge, MA.. Briefly, samples were added to wells that were pre-coated with anti-TNF antibody, in a 96-well plate. Biotinylated anti-TNF antibody was added 30 min later, and the plate was incubated for 2 h at room temperature. The plate was rinsed and streptavidin was added for 30 min. Enzymatic colour reagent was added and plates were developed in the dark. Stop solution was added, and the plate was read immediately with an ELISA reader set to 450 and 550 nm. Values from 550 nm were subtracted from values obtained at 450 nm. Values of controls were plotted on a standard curve and sample values were read from the curve. 2.5. Virus Four isolates of ASF virus were used for this study; these isolates were kindly provided by Dr. P. Wilkinson, IAH, Pirbright, UK. They were KWH Žmoderately virulent., Malawi Žhighly virulent., Perpignan Žalso highly virulent., and CV-1 Žavirulent in pigs.. Each of the virulent isolates had been passaged at least once in pigs, to provide a spleen or lymph node extract of ‘in vivo stock’ virus. The Perpignan virus was also available as an infectious blood preparation: lysed whole blood collected from an animal with acute clinical symptoms of ASF. Additional ‘in vitro stocks’ of these viruses were prepared by passaging them twice in cultured swine macrophages
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Žincubation temperature 378C., at input multiplicities of 0.001 immunofluorescence focus-forming units ŽIFFU. per cell. The CV-1 strain had been adapted to and passaged in CV-1 cells, and finally in swine macrophages Žincubation temperatures 378C.. Titres were expressed in IFFU per milliliter. The multiplicity of infection Žm.o.i.. for experiments was 0.5–1.0 IFFU per cell.
tein VP32, and an FITC-labelled isotype-specific conjugate. Anti-VP32 was diluted 1r1000, and was the gift of Dr. F. Alonso, CISA-INIA, Madrid, Spain. The conjugate was diluted 1r50 and was obtained from Southern Biotechnology, USA. Analysis employed a Becton Dickinson FACScan flow cytometer and Lysis II software.
2.6. Detection of Õirus infection
3. Results
Infected or uninfected control cells were removed from culture flasks by scraping at 24 h post-infection, and were fixed. Fixation was performed using a cell fixation and permeabilisation kit according to the manufacturer’s instructions ŽHarlan Sera-Lab, Sussex, UK.. They were labelled for flow cytometry using a monoclonal antibody against ASF virus pro-
3.1. Effect of temperature on size and granularity of cells Statistics from flow cytometer forward- and sidescatter plots were used to compare size and granularity of cells maintained at the two temperatures. No consistent differences in cell size were observed over
Fig. 1. Comparison of the size Žforward scatter, FSC, x-axis. and granularity Žside scatter, SSC, y-axis. of porcine monocyte-derived macrophages maintained at 378C Ža, c. or 39.28C Žb, d., either untreated Ža, b. or stimulated for 24 h with 1 m grml LPS Žc, d..
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Fig. 2. Phenotype of the monocytes isolated from porcine peripheral blood and used for the generation of macrophages in the experiments shown in the other figures. Ža. Percentage of cells Ž y-axis. clearly positive for the cell surface marker Žshown on the x-axis.. Žb. Relative intensity of fluorescence staining Ž y-axis. for each of the markers Žshown on the x-axis.. The values represent the mean from three different analyses Ždifferent preparations of blood monocytes. " SEM.
the course of various experiments. However, macrophages that had been incubated at 39.28C were more granular than cells maintained at 378C. Fig. 1a and b shows that granularity in unstimulated macrophages was higher in cells kept at 39.28C. Similarly, granularity was higher in cells incubated at the higher temperature and stimulated with 1 m grml of LPS ŽFig. 1c and d.. LPS-stimulated cells were examined 24 h post-stimulation. These experiments were performed three times; unstimulated cells kept at 398C were approximately 12% more granular than cells kept at 378C, and LPS-stimulated cells
kept at 398C were approximately 9% more granular than stimulated cells kept at 378C. 3.2. Effect of temperature on cell phenotype Cells were isolated as described in Section 2, plated in 75 cm2 plastic culture flasks, and incubated for 3 days at 378C or 39.28C, to allow differentiation into macrophages. They were removed from culture flasks at 3 days post-plating by scraping in PBS containing sodium azide, and labelled with monoclonal antibodies against MHC classes I and II,
Fig. 3. Expression of MHC class II on porcine monocyte-derived macrophages incubated at 378C or 39.28C. Ža. Percentage of cells Ž y-axis. recorded as MHC Class II positive; Žb. the relative fluorescence intensity Ž y-axis. of MHC class II expression on the positive cells. The x-axis shows the experiment number which generated the results.
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Table 1 Fluorescence intensity per cell a of the expression of MHC Class I, CD11ar18 and CD44 on porcine monocyte-derived macrophages maintained at 378C and 39.28C Cell surface antigen MHC Class I CD11ar18 CD44
Experiment 1 378C 39.28C
Experiment 2 378C 39.28C
Experiment 3 378C 39.28C
117 a 39 581
136 48 479
154 40 209
142 34 593
174 51 505
196 40 282
a Measurements were "10% coefficient of variation within experiments; for reference, the negative control labellings gave values -10.
CD11ar18, CD18, CD44 and the SWC3 myeloid marker. For comparison, the monocytes employed for the generation of these macrophages were similarly phenotyped ŽFig. 2.. These experiments showed that the expression of certain surface molecules was higher in cells incubated at 39.28C than in cells incubated at 378C. For example, Fig. 3a shows that a higher percentage of cells cultured at 39.28C expressed MHC class II, although the increase was slight in experiment number 5. Furthermore, the intensity of fluorescence per cell was generally higher in the cells maintained at 39.28C ŽFig. 3b.. Similarly, MHC class I fluorescence intensity was up-regulated in cells incubated at 39.28C. A summary of the results from three experiments are shown in Table 1. However, the 39.28C cells in experiment number 5 Žsee Fig. 3b. did not show a significant increase over the 378C cells. Values for the percentage of cells expressing the antigens shown in Table 1 are not shown because nearly 100% of cells kept at each temperature expressed these antigens. Table 1 also shows data for CD11ar18 and CD44 antigens. Fluorescence intensity values for these molecules did not significantly differ between cultures maintained at the two temperatures. Intensity values between cells maintained at the two temperatures and labelled with CD18 were variable Žresults not shown.. 3.3. Influence of temperature on production of tumour necrosis factor A porcine TNF ELISA was used as described in Section 2 to determine if temperature affected pro-
Table 2 Production of TNF-a is greater by macrophages maintained at 39.28C than at 378C Experiment
1 2 3 4
TNF-a Žpgrml. 378C
39.28C
0 0 51 41
106 145 118 51
The TNF was quantified in pgrml, and measured using a commercial ELISA kit for porcine TNF.
duction of TNF-a in stimulated macrophages. Cells isolated from the same animal were incubated at 378C or 39.28C. On the third day in vitro, they were stimulated with 1 m grml of LPS. Samples of the culture medium were removed at 24 h post-stimulation and stored at y708C until analysis. Table 2 shows levels of TNF-a produced in the cells during four experiments. TNF-a levels were very low or undetected in media from all cells maintained at 378C. However, the cytokine was detected in all of four media samples taken from cells incubated at the higher temperature. Thus, incubation at the lower temperature does not facilitate efficient TNF-a production by LPS-stimulated macrophages. 3.4. Effect of temperature on ASF Õirus replication Porcine mononuclear cells, isolated and incubated as described in the Section 2, were plated in 25 cm2 plastic culture flasks. At 3 days post-plating, they were infected simultaneously with the KWH isolate of ASF virus, at an m.o.i. of 0.5–1.0 IFFU per cell. Infected cells were detected flow cytometrically at 24 h post-infection, using the monoclonal antibody anti-ASFV VP32, as described in the Section 2. Cells from flasks maintained at different temperatures were fixed simultaneously. Fig. 4a shows data from four experiments. In each case, the percentage of antigen-positive cells in the 378C-cultures was higher than that in 39.28C-cultures. Furthermore, the difference in the proportion of infected cells between the two temperatures was nearly 40% in three out of four experiments. No differences in extracellular virus titre were observed between cultures maintained at the two temperatures Ždata not shown..
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Fig. 4. Replication of ASF virus in porcine monocyte-derived macrophages prepared and maintained at 378C or 39.28C. Cells were infected with 0.5 to 1.0 IFFUrcell, and analysed at 24 h post-infection for the presence of ASF virus-specific antigen ŽVP32. production Ž y-axis; ‘percentage infected cells’.. Ža. Infection with the moderately virulent KWH isolate of ASF virus; Žb. infections with the virulent Malawi ŽM. isolate, virulent Perpignan ŽP. isolate or cell-culture adapted avirulent CV-1 ŽCV. strain. The x-axis shows the number of the experiment which generated the results, and the virus used Žin panel b..
Infection detection experiments were repeated using three other isolates of ASF virus, in order to determine if in vivo virulence of the infecting virus would influence the outcome of the experiment. These isolates were Malawi, Perpignan, and CV-1. The m.o.i. and method for virus detection were identical to those used for the KWH isolate experiments. Each experiment with a separate virus was performed twice. As shown in Fig. 4b, more cells in the 378C cultures were antigen-positive at 24 h postinfection, regardless of the virus infecting them. Both the Malawi and Perpignan isolates were less efficient at producing antigen than CV-1 strain at 39.28C, compared to 378C. Such results could not be explained by the virus having been propagated in macrophage cultures at 378C. Both the in vitro propagated and the ‘in vivo’ stock of Perpignan virus— the latter prepared as lysed blood from an acutely infected pig—gave similar results to those in Fig. 4b. This finding implies that viruses may show differences in their abilities to replicate that are not evident when cells are maintained at 378C.
4. Discussion The body temperatures of most mammals are higher than 378C, and those of most birds, including chickens, are over 408C. Hypothermia in a human is
described as a drop in body temperature of 28C ŽTortora and Grabowski, 1993.. Thus, incubation of cells such as porcine cells, at 378C can result in the establishment of a mild hypothermic state. A wellknown characteristic of this condition is a decrease in cellular metabolism—including the rates of biochemical reactions—and a reduction in oxygen availability in tissues that results from the disruption of normal haemoglobin–oxygen dissociation ŽTortora and Grabowski, 1993; Radostits et al., 1994; Schubert, 1995.. The reduction in metabolic rate is approximately 5% to 7% per degree Celsius ŽReuler, 1978; Schubert, 1995.. All of these reductions would have a negative effect upon cellular function. The data presented in this paper show that a number of cell functions were reduced at 378C, notably the expression of MHC but not adhesion molecules, the production of TNF-a , and macrophage-tropic virus replication. The increased percentage of ASF virus antigenpositive cells at 378C may have been due to a number of factors. For instance, the increased granularity in the 39.28C cells implies that organelles such as lysosomes andror secretory vesicles may have increased in number or activity. A greater number of these organelles would mean an increase in antimicrobial enzymes available to the cells. Variations between the virus isolates in their optimal temperatures for replication have been observed
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with a number of viruses ŽAynaud et al., 1972; Hozinsky et al., 1966; Lwoff and Lwoff, 1960; MacKenzie, 1969.. For example, virulent isolates of classical swine fever virus replicated to highest titre in PK-15 cells maintained at 39.28C to 408C, while avirulent isolates preferred an environment of 338C to 348C ŽAynaud et al., 1972.. Isolates of low virulence replicated optimally at temperatures between those for the virulent and avirulent isolates ŽAynaud et al., 1972.. The authors of the study noted that the optimal replication temperature of the virulent isolates corresponded to the body temperature of a domestic pig. These observations imply that ASF virus might also replicate preferentially at higher temperature. However, the present study showed that ASF virus VP32 antigen production was detected in fewer cells from the 39.28C cultures at 24 h post-infection, but there was no difference in extracellular virus titre obtained from cultures maintained at the two temperatures. These observations are in contrast to those for classical swine fever virus. They would imply that antigen production is more effective at the lower temperature but virus maturation and assembly could be most efficient at the body temperature of the pig. An explanation for these apparently contradictory results may lie in the type of cells used to propagate the viruses. The virus isolates used in the classical swine fever study were all cultivated in PK-15 cell lines that had been passaged repeatedly at 378C. Such multiple passaging of the cells may have caused them to adapt to the lower temperature, and certain critical functional capabilities at the higher temperatures might have been impaired, allowing the virulent isolate to replicate to a higher titre. Alternatively, the increase in temperature to 39.28C may have allowed certain functions to increase in the cells. Furthermore, PK-15 cells are not intrinsically destructive, as are macrophages. Finally, the virulent classical swine fever virus isolates may, as the authors suggested, have been better suited to replicate at the temperature corresponding to the susceptible animal, at least in terms of infectious progeny production. ASF virus may also be better suited to replicate at the temperature of the susceptible animal. The data presented in this study showed that primary cultures of porcine macrophages function differently at 39.28C compared to 378C: they were
more granular, and they produced more TNF-a upon stimulation. These factors may explain why less ASF virus antigen was detected at 24 h post-infection: macrophages specifically destroy invading pathogens, and the culture of porcine macrophages at a temperature that is more than 28C lower than that to which they have adapted may well impair some of their functionality. This fact is of crucial importance to the study of the biological and biochemical characteristics of cultured cells: the use and study of cells maintained at a sub-optimal temperature will, in all likelihood, result in the generation of misleading results. These results may be related to pathogen infection, cytokine production, enzyme function, drug function, protein synthesis, production of new cell lines, or any number of parameters. The production of TNF-a by the cells used in this study illustrates this point elegantly. Cells maintained at 378C produced little or no TNF-a , in sharp contrast to cells incubated at 39.28C. Thus, incubation of cultured cells at the temperature of their donor species is a vital factor in their culture, and analytical applications.
Acknowledgements This work was supported by a Swiss Nationalfonds grant, number NF 31-33646.92. We wish to thank Rene´ Schaffner for technical assistance, and Dr. Christian Griot for interesting discussions.
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