Immunology 1979 37 429
Alveolar macrophages II.
INHIBITION OF LYMPHOCYTE PROLIFERATION BY PURIFIED MACROPHAGES FROM RAT LUNG
P. G. HOLT Children's Medical Research Foundation, Princess Margaret Hospitalfor Children, Subiaco, Western Australia, and Department of Microbiology, University of Western Australia
Received 12 October 1978, acceptedfor publication 14 November 1978
Summary. Macrophages were prepared from the lung, peritoneal cavity and blood of normal, unstimulated rats from a number of strains. The macrophages were purified by adherence, and characterized via surface markers, enzyme activity and phagocytic capacity, and subsequently tested for activity in cultures of mitogen-stimulated syngeneic lymphocytes. Peritoneal macrophages and blood monocytes were mildly stimulatory, or ineffective in modulating mitogeninduced DNA synthesis; peritoneal macrophages reconstituted the blastogenic responses of macrophage-depleted lymph node cell cultures to normal limits. In contrast, alveolar macrophages were markedly inhibitory to lymphocyte proliferation; in some instances inhibitory activity was demonstrable when added alveolar macrophages comprised only 004% of the total cells in culture. Lymphocyte proliferation induced by T-cell mitogens was more susceptible to this inhibition than was proliferation induced by the B-cell mitogen LPS. Alveolar macrophages recovered from SPF rats, while less in number, exhibited comparable inhibitory activity. These results form part of an emerging picture of the normal alveolar macro-
phage as a potential 'suppressor' of T-cell activity in the lung.
INTRODUCTION The traditional view of immunology emphasizes the importance of specific mechanisms in protection of the mammalian host against pathogens. It is becoming increasingly evident, however, that in many circumstances ultimate regulation of specific immunological processes may depend upon the activity of the least specialized elements of the immune system, the macrophages. The ability of these cells to non-specifically regulate lymphocyte proliferation, and their capacity to exert both positive and negative influences in response to different stimuli (reviewed by Nelson, 1976), places the macrophages in a unique position as potential regulators of variety of immunological phenomena. Much of what is known of the role of macrophages in immune processes stems from studies on cells derived from the peritoneal cavity, often in the form of induced exudates. It is now recognized that many of the properties of exudative macrophages differ considerably from those of unstimulated (resting) cells (Nelson, 1976; Wing & Remington, 1977; Keller, 1975; Baird & Kaplan, 1977), and consequently much of the
Correspondence: Dr P. G. Holt, Childrens Medical Research Foundation, Princess Margaret Hospital for Children, Subiaco, Western Australia. 00 19-2805/79/0600-0429$02.90 © 1979 Blackwell Scientific Publications
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P. G. Holt
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published literature in this area requires re-evaluation. Further, it is becoming evident that the supposedly 'unspecialized' macrophage may not represent a single, functional entity. The observations of significant differences between macrophage populations from different sites (Walker, 1976) suggests instead that macrophages may further differentiate in tissues along pathways largely determined by local micro-environmental stimuli, and as a consequence may express functions peculiarly suited to their particular site in the body. The situation of particular interest to this laboratory relates to macrophages on the air-side of the lung, a site wherein the breakdown of mechanisms which control immunological reactions leads to a variety of hypersensitivity diseases of largely unknown aetiology. The role of the resident (alveolar) macrophage population in locally arising immune responses is illdefined, and the extrapolation of information derived from studies of macrophages from other organs is not justified in the absence of direct proof to the contrary. Indeed, a number of authors have pointed to major differences between the activity of alveolar macrophages and those from other sources (Pavillard, 1963; Rhodes, 1975; Roseman, 1972; Dutton, 1976; Hunninghake & Fauci, 1976), and it has been suggested that one of the roles of macrophages in the lung may be to inhibit the expression of local T-cell dependent immune responses (Mackaness, 1971; Truitt & Mackaness, 1971). The present study examines the capacity of alveolar macrophages from the rat to influence the mitogeninduced proliferation of T and B lymphocytes in vitro. The data indicate that alveolar macrophages from unstimulated rats are uniquely suppressive to lymphocyte blastogenesis.
MATERIALS AND METHODS
Experimental animals Conventional rats of the WAG and Sprague-Dawley strains were obtained from the Perth Medical Centre Animal Breeding Unit. WAG rats were also obtained from the Royal Perth Hospital Research Centre, Western Australia. Specified pathogen-free (SPF) rats of the DA strain were supplied by Dr J. Smith, John Curtin School of Medicine, Australian National University, Canberra, Australia. Materials Petri dishes and microtest tissue culture plates (96
well) were from Falcon Plastics (U.S.A.), Medium RPMI 1640 was obtained from Gibco (U.S.A.), and foetal calf serum (FCS) was supplied by Commonwealth Serum Laboratories, Melbourne, Australia. Cannulae equipped with Luer fittings (employed for endobronchial lavage) were Bardic I-catheters (C. R. Bard International Ltd, Essex, U.K.).
Macrophage collection and purification Alveolar macrophages were collected by endobronchial lavage, employing pre-warmed (370) RPMI 1640 supplemented with 10% FCS and containing 12 mm lignocaine hydrochloride. Each lung was lavaged in situ three times with 7-10 ml fluid. The cells were washed in lignocaine-free medium. Single cell suspensions containing alveolar macrophages were incubated in petri dishes for 1 5-3 h at 370, non-adherent cells vigorously washed from the monolayers, and adherent cells detached via a further 30 min incubation in the presence of RPMI + 10% FCS + 12 mm lignocaine. The cells were washed and resuspended in FCSsupplemented RPMI, and viability assessed by trypan blue exclusion. Peritoneal macrophages were obtained via lavage, employing the medium above, and purified by adherence. Mononuclear cells were prepared from whole blood by Ficoll-Hypaque sedimentation, washed, and incubated from 1 5 to 3 h in petri dishes. Adherent cells (putative blood monocytes) were detached with lignocaine. This agent has previously been shown to reversibly detach macrophages in vitro without loss of viability (Rabinovitch & De Stefano, 1975). Characterization of macrophages Cells were incubated for 2 h in serum-supplemented RPMI containing colloidal gold (50 pg/ml). The cells (on glass coverslips) were subsequently stained with methyl green, and percentage phagocytic cells determined by observation under high magnification. Nonspecific esterase staining was performed employing the method described in Weir (1978). The percentage of cells containing Fc receptors for cytophilic antibody was determined employing the EA-rosette method described by Kedar, Ortiz de Landazuri & Fahey (1974). Preparation of lymphocytes Splenic and mesenteric lymph node lymphocytes were prepared as single cell suspensions from finely chopped tissues, and sieved through absorbent cotton wool. Thoracic duct lymphocytes were collected over a 16 h period into chilled phosphate-buffered saline
Alveolar macrophages II (PBS), via an indwelling catheter. Peripheral blood lymphocytes were prepared by Ficoll-Hypaque sedimentation. All cells were washed in medium containing serum, and viability assessed with trypan blue. Macrophage depletion of lymphocyte preparations was effected by glass-wool adherence according to the method of Folch & Waksman (1974).
Lymphocyte blastogenesis 2-0 x l0s mononuclear cells were dispensed into wells of microtest plates (Falcon Plastics, U.S.A., No. 3040) in 200 Pl RPMI (supplemented with glutamine) containing 10% FCS. Macrophages were added in aliquots of 10 gil. Mitogens were subsequently added in 10 pl volumes, at concentrations previously determined to elicit maximal blastogenic responses: PHA 50 pg, concanavalin A 0 5 gig, and LPS 10 Mg per well. The cultures were incubated at 370 in an atmosphere of 5% CO2 in air. Twenty-four hours later each well received 0 5 ,Ci titrated-thymidine. After a further 24 h incubation, the plates were processed, employing a Skatron Automatic Cell Culture Harvester (Skatron, Norway), and [3H]-DNA synthesis determined by liquid scintillation counting. Data were expressed as counts per minute (c.p.m.) per culture. RESULTS The characteristics of adherence-purified alveolar and peritoneal macrophages employed in these experiments are shown in Table 1. The criteria applied were activity of macrophage non-specific esterase, presence of Fc receptors, phagocytic activity and morphological characteristics. By these criteria, alveolar macrophage preparations were (on average) 96% pure, while those from the peritoneal cavity were 87% pure. The
Table 1. Characteristics of purified macrophage populations from WAG rats Marker Source of
macrophages Esterase Fc receptor Phagocytosis Morphology Lung Peritoneal
92-99
79-94
91-97
93-96
cavity
81-92
82-94
72-89
81-88
Data shown reflect the range of figures obtained from examination of adherence-purified macrophage populations from eight rats. The figures indicate the percentage of cells positive by the criteria shown.
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most notable differences between the two populations were cell size (alveolar macrophages were three to four times larger) and phagocytic activity; on average, 95% of the alveolar macrophage preparation took up colloidal gold, whereas the figure for the peritoneal population was 77%. Table 2 compares the activity of macrophages from the peripheral blood, the peritoneal cavity and the lung, in cultures of mitogen-stimulated syngeneic splenocytes. Alveolar macrophages were unique in their suppressive activity. When these macrophages were added in amounts equivalent to 10% of total cells in culture, mitogen-induced [3H]-DNA synthesis was completely inhibited, and inhibitory activity was invariably demonstrable down to alveolar macrophage: lymphocyte ratios of 1: 200. In contrast, mild inhibitory activity was (inconsistently) demonstrable with peritoneal macrophages and blood monocytes only at the highest concentrations employed. Table 3 indicates that the suppressive activity of alveolar macrophages is readily demonstrable with all lymphocyte populations from syngeneic animals. Table 4 examines the activity of rat alveolar macrophages in cultures of macrophage-depleted lymph node cells. As reported by Folch, Yoshinaga & Waksman (1973), rigorous depletion (3 h glass-wool adherence) of macrophages from lymph node preparations diminishes their response to mitogens, and the addition of purified peritoneal macrophages restores the response. Alveolar macrophages do not restore the capacity of these lymphocytes to respond to mitogens. Less rigorous depletion (1 h glass-wool adherence) increases PHA-induced blastogenesis, perhaps (as suggested by the studies of Folch & Waksman (1974) via removal of adherent T suppressors. The addition of syngeneic peritoneal macrophages did not alter PHA-induced responses in these cultures, whereas alveolar macrophages were again highly suppressive. Table 5 examines the possibility that the adherencepurification step employed during alveolar macrophage preparation may have selected a subpopulation of vigorously adherent, suppressive cells from the starting population. The data indicate only subtle differences between the activity of pooled lung washings, and subsequently adherent and nonadherent alveolar macrophages, which argues against this possibility. All rats examined in this study (in excess of 200) yielded alveolar macrophages which were suppressive towards lymphocyte blastogenesis; a range of activities was noted within the strain most studied (WAG),
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P. G. Holt Table 2. Comparative effects of WAG rat macrophages from the blood, peritoneal cavity and lung on mitogen-induced DNA synthesis in syngeneic spleen cells
Peritoneal macrophages
Blood monocytes
Alveolar macrophages
Number added macrophages*
PHA
Con A
PHA
Con A
PHA
Con A
0 2x 104 1 X 104 5 x103 2-5 x 103 1l25 x 103 6 25 x 102
47,653 30,421 48,520 65,217 67,158 53,521 49,127
32,517 21,523 38,614 47,519 51,423 38,525 31,476
48,125 40,742 51,586 50,724 48,416 47,643 47,917
31,250 30,346 46,417 46,116 50,412 40,411 39,433
47,724 510 1,125 2,517 11,215 19,763 38,426
33,321 717 914 3,476 17,423 34,426 30,173
Data shown are mean c.p.m. per culture; each well received 50 pg PHA or 0-5 Pg Con A. Background c.p.m. were below 500 c.p.m. and were not significantly affected by the addition of macrophages. All macrophages were lignocaine-treated during preparation.* Cultures contained 2 x 105 mononuclear cells-before the addition of macrophages.
Table 3. Comparative effects of WAG rat alveolar macrophages on blastogeneic (PHA) responses in lymphocytes from various sources Source of lymphocytes Number added macrophages Spleen Lymph node Thoracic duct Peripheral blood 0 2 x 104 1 x 104 5 x 103 2 5 x 103 1l25 x 103
13,541 500 422 3,621 8,817 14,020
31,111 199 325 790 9,125 16,426
66,291 561 811 6,557 18,629 32,099
37,941 190 2,131 9,689 25,281 26,817
Data shown are mean c.p.m. per culture; adherence-purified (96%) alveolar macrophages were used. All background counts were below 500 c.p.m. Table 4. Relative effects of rat peritoneal versus alveolar macrophages on PHA-induced blastogenesis in cultures of syngeneic macrophagedepleted lymph node cells Pre-treatment of LNC
Control
Glass wool, 1 h Glass wool, 3 h
Number of added
macrophages 0 2x 104 1 X 104 5x 103 2-5 x103 1l25x 103
PM*
AMt
PM
AM
PM
AM
35,527 34,612 59,726 61,222 16,214 15,297 595 32,126 1,250 50,813 2,152 25,317 43,125 1,976 61,257 5,186 38,526 1,720 45,916 2,843 66,515 6,791 41,257 1,953 51,257 6,125 71,242 12,116 39,423 3,146 41,376 9,742 63,426 23,111 24,714 5,325
Mesenteric lymph node cells from WAG rats were incubated in packed glass wool columns for 1 or 3 h, prior to plating into wells of PHA plates with varying numbers of macrophages; untreated lymph node cells served as controls. Data shown are mean c.p.m. per culture; background (unstimulated) counts were below 600 c.p.m. * Alveolar macrophages. t Peritoneal macrophages.
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Alveolar macrophages II Table 5. Effect of fractionated and unfractionated alveolar macrophages from WAG rats on PHA-induced DNA synthesis in cultures of syngeneic spleen cells
Source of alveolar macrophages Number added macrophages Pooled lung washings* Adherence purifiedt Washings from monolayert 0 2 x 104 1 x 104 5 x 103 2-5 x 103 125 x 103
29,516 3,475 5,613 11,267 21,465 31,246
30,250 2,620 4,125
31,252 855
3,120 7,956 16,451 23,251
8,525
20,561 31,151
Data shown are mean c.p.m. per culture. Background (unstimulated) counts averaged 320; the addition of macrophages increased counts up to 510 c.p.m. * Pooled washings from two rats; comprised 77% macrophages. t The pooled washings were plated out for 2 h, washed vigorously, and cells from the resulting monolayer detached with lignocaine; comprised 97% macrophages. t These were the cells washed off the monolayers, prior to the addition of lignocaine; comprised 68% alveolar macrophages.
and the extremes of this range are depicted in Fig. 1. Rat number 1 reflects the top end of this spectrum (approximately 20% of animals), where the addition of as few as eighty alveolar macrophages is sufficient to markedly suppress the PHA-induced blastogenic response of 20 x 105 splenocytes. Rat number 2 is indicative of the opposite extreme, where a minimum of 5-0 x 103 alveolar macrophages is required to elicit comparable suppression. Figure I also draws comparisons between the relative suppressive activity of rat alveolar macrophages towards T-cell (PHAinduced) and B-cell (LPS-induced) blastogenesis. While alveolar macrophages could be shown to suppress B-cell blastogenesis, suppressive activity was consistently greater against T-cell responses (see particularly Rat 1). In many experiments, much greater differences than those shown in Fig. 1 were observed, and suppression of LPS-induced blastogenesis was often demonstrable only at the highest macrophage concentration. The experiments of Table 6 address questions of strain differences, infection status, and non-specific environmental influences. These controls are particularly pertinent to this study, given the known differences between the yield of alveolar macrophages from SPF and conventional animals (Rylander, 1974) and the exquisite sensitivity of alveolar macrophage populations to non-specific environmental stimuli (reviewed by Holt & Keast, 1977). The SPF-rats employed yielded less than 30% of the alveolar macro-
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0
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004015 06 25 50 2 008 03 Added mocrophages (/ culture)
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Figure 1. Range of inhibitory activity observed in alveolar macrophages from WAG rats. Data shown indicate percentage inhibition of mitogen-induced DNA synthesis in spleen cells, employing alveolar macrophages from two different rats (circles, rat 1; squares, rat 2). Numbers of added macrophages were between 2 x 104 (10% of cells in the wells) and 80 (i.e. 0.04%). Cultures were stimulated with 50 pg PHA-P (closed symbols) or 10 pg LPS (open symbols).
phages recoverable from conventional rats (data not shown). Nevertheless, the activity of their cells were comparable to those seen with macrophages from conventional animals of two other rat strains (Table 6). Alveolar macrophages of WAG rats from Supplier No. 2 appeared more suppressive than those from Supplier No. 1; a mycoplasmal infection (lung) is endemic in the former colony, and may be responsible for the increased suppressive activity.
P. G. Holt
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Table 6. Effect of variations in rat strain, infection status, and supply source on activity ofalveolar macrophages in PHA-stimulated cultures of syngeneic spleen cells
Conventional WAG strain SPF DA-strain
Conventional Sprague-Dawley strain
Supplier No. I Supplier No. 2
Number added
macrophages 0 2 x 104 1 X 104 5 x103 2-5 x 103 1 25 x 103
PHA Con A
PHA
Con A
PHA Con A PHA Con A
31,652 14,230 252 1,834 315 7,366 759 14,380 18,240 4,040 24,888 10,466
37,991 1,481 2,602 5,997 12,976 18,627
29,176 426 691 1,121 9,231 12,227
37,941 25,267 47,256 41,257 711 255 521 195 824 726 2,130 1,976 891 9,687 4,230 1,125 25,225 14,252 7,252 3,217 26,815 22,263 9,573 6,171
Adherence-purified alveolar macrophages (minimum purity 95%) were prepared from conventionally housed male Sprague-Dawleys, specified pathogen-free animals of the DA strain, and conventional WAGs from two different animal houses (supplier No. 2 WAG colony has lowgrade mycoplasma endemic). These macrophages were titrated into cultures of syngeneic spleen cells as shown. Data shown are mean c.p.m. per culture; background counts varied between 180 and 745 c.p.m.
DISCUSSION It is generally accepted that normal peritoneal macrophages can serve as obligatory accessory cells in a wide range of cellular immune reactions, including mitogen- and antigen-induced T-cell blastogenesis; the role of 'excess numbers' of macrophages in lymphocyte cultures is less clear. A variety of studies suggest that inhibition may result from the inclusion of high numbers of normal peritoneal macrophages in lymphocyte cultures undergoing blastogenesis (Parkhouse & Dutton, 1966; Harris, 1965; Waldman & Gottlieb, 1973; Keller, 1975), MLC reactions (Fernbach, Kirchner and Herberman, 1976) or induction of antibody synthesis (Hoffman, 1970). In reviewing the aforementioned literature, Wing & Remington (1977) have suggested that this dichotomy may be reconciled by the adoption of a more rigorous definition of what constitutes a 'normal' macrophage population, viz. those where contaminating cells constitute less than 5%, and those which have been collected from normal animals not pretreated with macrophage-inducing agents. Under these circumstances, pure populations of unstimulated peritoneal macrophages clearly do not suppress T-cell blastogenesis, even when present in high numbers (Wing & Remington, 1977). The results of the present experiments with blood monocytes and peritoneal macrophages from the rat (Table 2) are consistent with the results of Wing & Remington (1977), and indicate that both of these
macrophage populations from normal animals are not suppressive to T-cell blastogenesis. Alveolar macrophages from the same animals, however, obtained in highly purified form (>95%) were strongly suppressive to PHA- and Con-A-induced blastogenesis. These results form part of an emerging picture of the 'normal' alveolar macrophage as a functionally distinct, perhaps unique, member of the mononuclear phagocyte system. Direct comparisons between alveolar and peritoneal macrophages have yielded the following observations: while exhibiting comparable phagocytic capacity and markedly increased activity of lysosomal hydrolases, alveolar macrophages are considerably less efficient in bacterial killing (Pavillard, 1963); they are considerably more active in PHAand antibody-induced cellular cytotoxicity (Hunninghake & Fauci, 1976); Fc receptor activity is diminished (Rhodes, 1975) as is their capacity to yield immunogenic antigen (Cohn, 1964) or RNA (Fishman & Adler, 1970) after antigen ingestion; and they cannot serve as accessory cells in the in vitro induction of antibody synthesis (Roseman, 1972; Dutton, 1976). Taken together with the results of the present experiments wherein alveolar macrophages were demonstrated to actively suppress lymphocyte proliferation, it may be suggested that the resident macrophage population on the air-side of the respiratory tract contributes towards the maintenance of a microenvironment which severely limits the local induction and expression of T-cell dependent immune responses.
Alveolar macrophages II This possibility was suggested earlier by Mackaness (Mackaness, 1971; Truitt & Mackaness, 1971) in studies of the pathogenesis of Listeria infections in the lungs of mice. The latter studies also suggested that recruitment of blood monocytes into the lung as a result of inflammation effectively overcame local inhibitory barriers, and permitted the expression of T-dependent immunity. The marked differences observed here between the activity of blood monocytes and alveolar macrophages suggest a potential mechanism for the model proposed by Mackaness, and may also underlie the increased capacity of the lung to respond immunologically to antigenic challenge which accompanies inflammation (Matsamura, 1970; Nettesheim & Williams, 1974; Peterson, Thrall, Moore, Stevens & Abramoff, 1977). This possibility is currently under investigation in these laboratories. In assessing the biological implications of these data, it is necessary to define the mechanism(s) underlying the cytostatic activity of alveolar macrophages, and further to examine the activity of these cells in a number of other species. The question of mechanism is addressed in the accompanying paper (Holt, 1979). ACKNOWLEDGMENTS This work was supported by Princess Margaret Children's Medical Research Foundation. The author thanks Dr G. Mayrhofer for the supply of TDL cells, Dr Mayrhofer and Professor K. J. Turner for helpful comments and criticism, and Miss J. Harvey for technical assistance. REFERENCES BAIRD L.G. & KAPLAN A.M. (1977) Macrophage regulation of mitogen-induced blastogenesis. I. Demonstration of inhibitory cells in the spleens and peritoneal exudates of mice. Cell Immunol. 28, 22. COHN, J.A. (1964) The fate of bacteria within phagocytic cells. III. Destruction of an Escherichia coli agglutinogen within polymorphonuclear leucocytes and macrophages. J. exp. Med. 120, 869. DUTTON R.W. (1976) Personal communication cited by Kaltreider, H.B., in Immunologic and Infectious Reactions in the Lung, p. 84, Marcel Dekker Inc., New York. FERNBACH B.R., KIRCHNER H. & HERBERMAN R.B. (1976) Inhibition of the mixed lymphocyte culture by peritoneal exudate cells. Cell Immunol. 22, 399. FISHMAN M. & ADLER F.L. (1970) In: Mononuclear Phagocytes (Ed. by R. van Furth), p. 581. Blackwell Scientific
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