Macrophage Cytophilic Antibody in Mice - Infection and Immunity

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Jun 4, 1971 - Michael, J. G. 1966. The release of specific antibacterial ... Neuberger and E. L. Tatum (ed.), North Holland Research. Monographs, vol. 11.
Vol. 4, No. 4 Printed in U.S.A.

INFECTION AND IMMUNITY, Oct. 1971, p. 402-406

Copyright @ 1971 American Society for Microbiology

Macrophage Cytophilic Antibody in Mice: Mechanism of Action of Bacterial Lipopolysaccharide on the Uptake of Immunoglobulins by Mouse Peritoneal Cells IAN R. TIZARD' Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada

Received for publication 4 June 1971

The action of injected bacterial lipopolysaccharide upon the uptake of cytophilic antibodies by macrophages was found to be unrelated either to changes in surface charge or to the rate of anaerobic glycolysis of these cells. It is apparently mediated by alterations in the levels of material in the serum which enhances the uptake of cytophilic antibody by macrophages. This enhancing material does not appear to be an immunoglobulin nor can it be generated by the action of endotoxin on either whole blood or serum.

Endotoxins produce a very wide range of physiological and pathological effects in susceptible animals. Although many of these effects are biphasic in nature, very few of them have been shown to be related. The response of the macrophage receptor for cytophilic antibodies to bacterial lipopolysaccharide is no exception. It stands as an isolated phenomenon with no apparent relationship to any of the other responses induced by extracts of cell walls of gram-negative bacteria. It was found that lipopolysaccharide from Escherichia coli when injected into normal mice produced changes in the surface properties of the peritoneal macrophages of these animals, so that their ability to adsorb macrophage cytophilic antibodies was altered (12). After a single intravenous injection of this lipopolysaccharide, a triphasic sequence of changes in macrophageadsorptive capacity occurred. Enhancement of antibody uptake took place 5 min after injection; however, this enhancement occurred for only a few minutes and was followed by a period of depressed uptake. Maximum depression occurred approximately 30 min after injection and preceded a prolonged phase of enhanced adsorption which reached a maximum 24 hr after injection before declining slowly. This second phase of enhanced adsorption was found to be inhibited or reversed either by multiple injections of 1 Present address: Animal Diseases Research Association, Moredun Institute, Edinburgh, U.K.

lipopolysaccharide or by prior immunization of mice with dead E. coli. The investigations reported in this paper were designed to identify the mechanism of altered uptake of cytophilic antibody by macrophages in the hope of relating these changes to other effects of lipopolysaccharide upon reticuloendothelial function (3) and also to provide information on the factors which influence the cellular fixation of immunoglobulins. MATERIALS AND METHODS Animals. White Swiss mice of both sexes, weighing between 20 and 25 g and reared in this laboratory, were used. Antigens and antisera. Sheep erythrocytes were obtained at weekly intervals from the defibrinated blood of a female Blackface sheep and stored at 4 C. Antisera were raised as described previously (12). Baterial lipopolysaccharide. Difco E. coli 026:B6 prepared by the method of Westphal and Luderitz (13) was stored as a stock solution of 200 ,ug/ml in phosphate-buffered saline (pH 7.2). Preparation of cell monolayers and titration of cytopkilic antibodies. These procedures were performed as described previously (12). Electrophoresis of peritoneal cells. Electrophoresis was performed in a cylindrical, all-glass, electrophoresis chamber by the method of Bangham ct al. (1). All solutions in contact with the electrophoresis chamber were made up in doubly glass-distilled water which had not been exposed to ion-exchange resin. Anaerobic glycolysis of peritoneal cells. The release 402

VOL. 4, 19 71

MACROPHAGE CYTOPHILIC ANTIBODY

of carbon dioxide from peritoneal cells was measured in a Warburg constant-volume respirometer under an atmosphere of 94.71% nitrogen and 5.29% carbon dioxide (Canox). Measurement of immunoglobulin G levels in serum. The levels of total immunoglobulin G (LgG) in mouse sera were measured by radial immunodiffusion by the method of Mancini, Carbonara, and Heremans (7). RESULTS Two of the changes reported to occur in cells

after administration of endotoxin were considered to be capable of influencing immunoglobulin adsorption. These changes were in the surface potential of the cell as reflected in its electrophoretic mobility (2) and in the metabolic activity of the cell as reflected in its rate of carbon dioxide production under anaerobic conditions (14). Electrophoretic mobility of lipopolysaccharidetreated mouse macrophages. Bergmann et al. (2) demonstrated that the electrophoretic mobility of rabbit polymorphonuclear neutrophils underwent a biphasic change after treatment of the animal with bacterial lipopolysaccharide. These cells showed a drop in electrophoretic mobility which reached a minimum value 2 hr after injection of lipopolysaccharide but subsequently increased rapidly to reach its maximum value 8 hr after injection, from which it slowly declined. It was considered improbable that similar changes in macrophage elecrophoretic mobility would directly affect immunoglobulin adsorption especially at a physiological pH. Nevertheless, changes in the degree of adsorption of more highly charged serum proteins could be expected to alter the amount of cell surface available for immunoglobulin uptake. The electrophoretic mobility of peritoneal macrophages from normal and lipopolysaccharide-treated mice was therefore determined (Table 1 and Fig. 1). No significant difference could be detected between normal macrophages and cells examined 30 min and 24 hr after administration of lipopolysaccharide. Anaerobic glycolysis of mouse peritoneal cells from lipopolysaccharide-treated mice. Woods et al. (14) demonstrated that mouse peritoneal cells underwent a triphasic series of changes in their rate of anaerobic glycolysis in response to a single injection of either Salmonella enteritidis or Shigella dysenteriae endotoxins. Although the time course of this reponse appeared to be unrelated to the changes occurring in cytophilic antibody uptake, experiments were performed to determine whether there is any relationship between these alterations in metabolic activity and changes in cytophilic antibody uptake. The anaerobic glycolytic activity of cells from

403,

TABLE 1. Electrophoretic mobility of peritoneal macrophages derived from normal and lipopolysaccharide-treated micea Cells

No.of

Mobility (uam per sec per v per cm)

100

1.49 ±fi 0.25 SD

25

1.36 :1 0.24 SD

examsined

Normal macrophages. Macrophages 30 min after lipopolysaccharide treatment.... Macrophages 24 hr after lipopolysaccharide treatment ............

100

1

.36 i 0.22 SD,

a Electrodes, reversible Ag/AgCl/KCl; Po-tential difference, 50 v; current, 0.017 ma. Diluent,. 0.145 M NaCl containing 3 X 104 M NaHCO . Electrical tube length, 126.5 mm; diameter, 2.014¢ mm. Distance timed, 40.3 Mm. Dose of lipopolysaccharide, 1.0 jig per g of body weight. SD, standard deviation.

20 male Swiss mice of comparable age and size was examined at intervals after each had received a single intravenous dose of E. coli lipopolysaccharide which ranged from 0.5 to 2,g per g of body weight. The release of carbon dioxide was measured as described above. Under these conditions, no relationship could be detected between macrophage metabolic activity as measured in this way and the degree of enhancement or inhibition of cytophilic antibody uptake expressed as a percentage of the normal value (Fig. 2). The correlation coefficient r between these two factors was 0.0004. The average rate of CO2 production under anaerobic conditions for normal mouse peritoneal cells was found to be 1.65 ,liters of CO2 per 106 cells per hr. Changes in the properties of mouse serum accounting for alterations in the capacity of mouse peritoneal cells to adsorb cytophilic antibodies. The ability of a macrophage to adsorb cytophilic antibodies when these antibodies are in relative excess depends upon the number of available receptors upon the cell surface. The number of these free receptors will in turn be influenced by exposure to cytophilic immunoglobulins in vivo.

Cells derived from an environment with a high concentration of cytophilic antibodies will have fewer available receptors when tested in vitro. Investigations were therefore performed to determine whether injected lipopolysaccharide influences cytophilic antibody uptake in vitro by its capacity to alter the serum levels of nonspecific cytophilic material or material which influences the uptake of cytophilic antibody by macrophages. The levels of such material were estimated by

404

INFEC. IMMUN.

TIZARD Normal cells.

% cells counted.

30 Cells from lipopolysoccharide treated animals.

20

-

10

-

10

0-

2.0

18

16

14

13

1'2

ll

Sec. to

pass

40O3 pm.

10

FIG. 1. Electrophoretic mobility ofperitonieal cells from normal anzd lipopolysaccharide-treated mice. For details, Table 1.

see

3.

Carbon dioxide p

rod uction

.pI/lo6cells/hr 2-

1

I

0

100

200

300

400

C/ocytophilic

antibody

uptake

FIG. 2. Relationship between carbon dioxide production of lipopolysaccharide-treated peritonieal cells unlder anaerobic conditions and their capacity to adsorb cytophilic antibodies. (Expressed as a percenltage of the ability of normal cells to take utp these anitibodies).

the capacity of the material to inhibit or enhance the uptake of cytophilic antibodies of known specificity. The serum to be tested for "nonspecific cytophilic" activity was adsorbed once with sheep erythrocytes and diluted 1:5 in Hanks basic salt solution. The capacity of this unknown serum to inhibit the absorption of cytophilic antibody to macrophages was then tested in a system in which a standard anti-sheep erythrocyte serum containing cytophilic antibodies was used. The degree of inhibition or enhancement of cytophilic antibody uptake was judged by alterations in the cytophilic antibody titer of the standard serum. It was found that serum taken from mice 30 min after injection of lipopolysaccharide markedly inhibited cytophilic antibody uptake by macrophages and that serum taken 24 hr after injection caused enhanced uptake.

It addition, it was found that a relationship existed between the degree of alteration in antibody absorption by cells from treated animals and the change induced in normal cells by serum from these treated animals [correlation coefficient r 0.689 (Fig. 3)]. Twenty individual mice were used in determining this relationship. Identification of enhancing and inhibiting material in mouse serum. Although it has been demonstrated that "nonspecific" cytophilic IgG may both inhibit (6, 9, 11) and enhance (11) specific cytophilic antibody uptake and although cytophilic antibodies may be released into serum by the action of bacterial lipopolysaccharide (5, 8, 10), experimental evidence suggests that IgG is not the major inhibitor or enhancing factor in lipopolysaccharide-induced changes in cytophilic antibody uptake. When IgG levels as measured by radial im=

MACROPHAGE CYTOPHILIC ANTIBODY

VOL. 4, 1971

Woods et al. (14) were able to alter the rate of macrophage metabolism in vitro by exposure to lipopolysaccharide. This, taken in conjunction with the lack of in vitro response of antibody receptors and the total lack of correlation between CO2 production under anaerobic conditions and cytophilic antibody uptake, indicates that the rate of cellular metabolism, as measured in this way, has no apparent influence upon the availability or avidity of macrophage antibody re-

300

%Change in antibody uptake by normal cells in the presence of serum from treated animals.

405

2000

ioc

100

ceptors.

200

300

400

The adsorption of macrophage cytophilic antibodies, as measured in vitro by the rosette test, %Change in antibody uptake by cells of treated animals. is influenced by the availability of macrophage FIG. 3. Relaitionshlip between the change in antibody receptors. These in turn, are affected by the uptake by norrmal macrophages in the presence of presence, either already on the cells or in the serum under test, of "nonspecific" cytophilic serum from 1'ipopolysaccharide-treated anzimals expressed as a p;ercentage of the nzormal value and the material. Such "material" may be cytophilic observed chanAge in adsorptive capacity of the cells antibodies directed against antigens other than from these tre ated animals also expressed as a per- the ones under test. Any changes in the level of such "nonspecific" cytophilic material will be cenitage of the normal value. reflected in the apparent uptake of specific munodiffusiol n in the serum of 20 lipopoly- cytophilic antibody. It has been demonstrated saccharide-tre ated animals were compared that lipopolysaccharide may induce the release of with the IgG levels in the sera of 20 untreated preformed immunoglobulins from bone marrow and spleen (5, 8, 10). It may reasonably be mice, no siggnificant differences were detected either 30 mi n or 24 hr after administration of assumed that such released material could conlipopolysacch iaride, at a time when there were tain cytophilic immunoglobulins and thus inmarked alter ations in the adsorptive capacity fluence specific cytophilic antibody adsorption. of their peritl oneal macrophages. In addition, 10 However, this is apparently not the primary gnotobiotic n lice were found to exhibit a triphasic mechanism of the triphasic response to lipopolyresponse to Ilipopolysaccharide similar in magsaccharide, since such a response may be detected nitude to theat shown by conventional animals in gnotobiotic mice. This is not, of course, abdespite undet ;ectable levels of immunoglobulin in solute evidence that immunoglobulins do not their serum. The serum of these lipopolysac- mediate the triphasic response. If, however, they charide-treate d mice was also capable of en- do participate in this reaction, it must be conhancing cyto philic antibody uptake by normal sidered, firstly, that they can do so in extremely macrophages when taken 24 hr after lipopoly- small quantities, and, secondly, since the triphasic response of gnotobiotic animals was of saccharide ad[ministration. Enhancing activity could not be generated in similar magnitude to that in conventional anieither serum c :r whole blood from conventional or mals, this indicates that the considerable difference gnotobiotic mice after exposure to lipopoly- in immunoglobulin levels between these two saccharide in vitro for 24 hr (0.02 mg of lipopoly- groups of animals had no significant influence saccharide wi is added to 0.5 ml of fresh serum or upon the response. 1 ml of whole blood). It is, however, apparent that a factor (or fac0

i

DISCUSSION From these investigations, it appears that the effect of bac terial lipopolysaccharide upon the uptake of cyt ophilic antibody by macrophages is unrelated to s several of the other effects produced by these comj )ounds upon macrophages. studies (12), it was known knw that ht From previ iOUS studies lipopolysacch aride did not exert a direct effect

pouns

(12),riwages

hage receptors. Lipopolysaccharide added to ma crophage monolayers in vitro has no apparente effect upon their capacity to adsorb cytophilic antibody. upon macrop

tors) is present in the serum of treated mice which

influences cytophilic antibody uptake. It is probably not immunoglobulin, it is not generated by addition of lipopolysaccharide to serum or whole blood, and it is probably not therefore related to any clotting factors or leukocyte pyrogens. A factor which is known to influence macrophag acivt in a ytpii phage activityin a similar way to cytophilic antibodies is macrophage migration inhibition factor. This is a member of a class of soluble factors released as a result of the interaction of antigen upon sensitized lymphocytes known as

.iiawyt

406

TIZARD

"lymphokines" (4). It is possible that the enhancing factor reported here also belongs to this group and that interaction of antigen (in this case bacterial lipopolysaccharide) and sensitized lymphocytes results in its release. However, there is no evidence for the occurrence of such a reaction, and classifying this enhancing factor as a "lymphokine" at the present time is entirely speculative.

INFEC. IMMUN.

mice. This investigation was supported by the Medical Research Council of Canada.

generated by lymphocyte activation. Nature (London) 224:38-42. 5. Hill, W. C., and D. Rowley. 1967. The origin of antibody released into serum following injection of bacterial lipopolysaccharide. Aust. J. Exp. Biol. Med. Sci. 45:693-701. 6. Jonas, W. E., B. W. Gurner, D. S. Nelson, and R. R. A. Coombs. 1965. Passive sensitisation of tissue cells. 1. Passive sensitisation of macrophages by guinea-pig cytophilic antibody. Int. Arch. Allergy 28:86-104. 7. Mancini, G., A. 0. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235-254. 8. Michael, J. G. 1966. The release of specific antibacterial antibodies by endotoxin. J. Exp. Med. 123:205-212. 9. Nelson, D. S. 1968. Macrophages and immunity. In A. Neuberger and E. L. Tatum (ed.), North Holland Research Monographs, vol. 11. North Holland, Amsterdam-London. 10. Rowley, D., and J. K. Turner. 1964. Increase in macroglobulin antibodies of mouse and pig following injection of bacterial

LITERATURE CITED 1. Bangham, A. D., R. Flemans, D. H. Heard, and G. V. H. Seaman. 1958. An apparatus for the electrophoresis of small particles. Nature (London) 182:642-644. 2._Bergmann, H., G. Buschman, P. Doering, E. Fritze, and F. Wendt. 1954. Der Einfluss bakterieller pyrogene (lipopolysacchaide) auf die phagozeaktivitat der granulozytes und auf die elektriscke oboflachen iodung menchlien blutzellen in vivo. Klin. Woschenschr. 32:500-503. 3. Biozzi, G., B. Benacerraf, and B. N. Halpern. 1955. The effect of Saim;. typhi and its endotoxin on the phagocytic activity of the reticuloendothelial system in mice. Brit. J. Exp. Pathol. 36:226-235. 4. Dumonde, D. C., R. A. Wolstencroft, G. S. Panayi, M. Matthew, J. Morley, and W. T. Howson. 1969. "Lymphokines" non-antibody mediators of cellular immunity

lipopolysaccharide. Immunology 7:394-402. 11. Tizard, I. R. 1970. Macrophage cytophilic antibodies in mice: the relationship between the adherence of antigen to macrophages mediated by macrophage-cytophilic antibodies and opsonic adherence antibodies. Int. Arch. Allergy 39: 201-209. 12. Tizard, I. R. 1971. Macrophage cytophilic antibody in mice: effect of bacterial lipopolysaccharide on the uptake of immunoglobulins by mouse peritoneal cells. Infec. Immun. 3:472-477. 13. Westphal, O., and 0. Luderitz. 1953. Uber die chemische und biologische analyse hochgereingter bakterienpolysaccharide. Deut. Med. Woschenschr. 78:17-19. 14. Woods, M. W., M. Landy, J. L. Whitly, and D. Burk. 1961. Symposium on bacterial endotoxins. III. Metabolic effects of endotoxins on mammalian cells. Bacteriol. Rev. 25:447456.

ACKNOWLEDGMENTS I thank G. A. Gresham for the use of his cell-electrophoresis apparatus and 0. P. Miniats for generous supplies of gnotobiotic