Unique determinants of alveolar macrophage spontaneous and chemokinetically stimulated migration

CELLULAR

IhlMUNOLOGY

Unique

39,

36-46

(1978)

Determinants

of Alveolar

and Chemokinetically JAN G.DOHLMAN Departnaozt

of Medicine,

Macrophage

Stimulated,

Spontaneous

Migration

AND EDWARD J. GOETZL~

Robert B. Brigham Hospitul and Harvard Boston, Massaclmsctts 02115 Received

Medical

School,

12,197s

January

The in vitro migration of rabbit alveolar and peritoneal macrophages was quantitated by an agarose well assay which permitted the distinction of chemokinetic and chemotactic patterns of stimulation by rabbit serum, tryptic fragments of the fifth component of complement, and the synthetic peptide formyl-methionyl-phenylalanylleucine. The peritoneal macrophages exhibited greater chemotaxis than the alveolar macrophages, but the magnitude of the chemokinetic response of both macrophage populations to each stimulus was much greater than that of the corresponding chemotactic response. Preincubation of macrophages with 2,4-dinitrophenol suppressed the spontaneous and chemokinetic migration of the alveolar macrophages without influencing the migration of the peritoneal macrophages, while iodoacetate inhibited the migration of both types of macrophages. The addition of a crude preparation of surfactant to the macrophages stimulated the migration of both the alveolar and peritoneal populations. Alveolar macrophages are thus not only uniquely adapted to the high oxygen concentrations in their environment, but may perform their surveillance of the pulmonary surfaces more efficiently as a result of the presence of surfactant or related lipoproteins.

INTRODUCTION The efficient surveillance of distal bronchopulmonary airways by alveolar macrophages, which occupy < 1% of the surface, is in part dependent on the spontaneous and stimulated migration of this specialized leukocyte population (1, 2). Some iw vitro .analyses of the functions of alveolar macrophages, as compared to peritoneal macrophages, have revealed unique biochemical and other characteristics which may be related to the adaptations of alveolar macrophages to their pultnonary environment. Substanti.al differences in the metabolic patterns of phagocytizing alveolar and peritoneal macrophages of guinea pigs have been found by direct measurements and the application of specific metabolic inhibitors (3). Alveolar macrophages exhibited more active aerobic metabolism of glucose than peritoneal macrophnges and the phagocytic capacity of the former was predominantly dependent on oxidative phosphorylation, as compared to glycolysis. In apparent contrast, one study of macrophage migration, utilizing the glass capillary tube assay, revealed com1Dr. Howard

Goetzl is Director Hughes Medical

of the Laboratories Institute.

for

36 OOOS-8749/78/0391-0036$02.00/O Copyright All rights

0 1978 by Academic Press, of reproduction in any form

Inc. reserved.

the

Study

of Immunological

Diseases

of the

DETERMINANTS

OF

ALVEOLAR

MACROPHAGE

MIGRATION

37

parable metabolic prerequisities for alveolar and peritoneal macrophages (4). As ouabain enhanced guinea pig alveolar macrophage migration from glass capillary tubes while inhibiting that of peritoneal macrophages (5), the two types of macrophages may also differ in some membrane transport systems or the coupling of such systems to migration. Although early studies with conventional micropore filter chambers did not demonstrate chemotactic migration of macrophages (6)) the use of very thin filters and brief incubation periods has permitted the recognition of macrophage chemotactic responses to several defined stimuli including peptides and lipids (7, 8). The agarose well assay is especially well suited to the comparative assessment of alveolar and peritoneal macrophage migration over intervals of 12 to 48 hr and it permits a complete analysis of the direction and magnitude of cell responses to each concentration of a stimulus. Thus chemokinetic enhancement, which is solely dependent on the concentration of the stimulus, can be readily distinguished from chemotaxis, which is also dependent on the concentration gradient of the stimulus. The application of the agarose well assay both permitted the recognition of a predominantly chemokinetic response of alveolar and peritoneal macrophages and defined differences in the metabolic prerequisites of the two populations of macrophages. METHODS

AND

MATERIALS

Plastic plates containing twenty-four 16 mm diameter cups (Costar, Cambridge, Mass.), Dulbecco’s phosphate buffered saline without calcium or magnesium and 2~ concentrated Medium 199 with Hanks’ balanced salt solution (Microbiological Associates, Walkersville, Md.), Bacto Certified gelatin (Difco Laboratories, Detroit, MI.), type HSA Litex agarose (Accurate Chemical and Scientific Corporation, Hicksville, N.Y.), 3 to 4 kg male albino rabbits (Margaret’s Home Farms, Greenfield, Mass.), synthetic dipalmitoyl phosphatidylcholine (Supelco, Inc., Bellefonte, Penn.), 2,4-dinitrophenol (Mann Research Laboratories, New York, N.Y.), iodoacetic acid (Eastman Kodak Co., Rochester, N.Y.), certified grade methanol and sodium acetate-buffered 10% ( v: v) formalin solution (pH 7.1) (Fisher Scientific Co., Medford, Mass.) were obtained as noted. C.5 was purified from 200 ml of human serum by sequential chromatography on TEAE-cellulose ( Cellex-T, Bio-Rad Laboratories, Richmond, Calif.), and hydroxylapatite (Hypatite C, Clarkson Chemical Co., Inc., Williamsport, Penn.) as described (9), and was quantitated by a standard radial immunodiffusion assay. C.5 fragments were generated by digestion of purified C5 for 15 min .at 37°C with trypsin that had been previously treated with diphenyl carbamyl chloride (Sigma Chemical Co., St. Louis, MO.) ; residual trypsin was inactivated by heating for 30 min at 61 “C (10). Formylmethionyl-phenylalanyl-leucine (F-Met-Phe-Leu) which had been synthesized and purified by Dr. Richard Freer was kindly supplied to us by Drs. Henry Showell and Elmer Becker. Rabbit serum was heated at 56°C for 30 min prior to use. Preparation of rabbit macrophages and sawfactant. Peritoneal macrophages were harvested 4 days after the instillation of 50 ml of extra heavy viscosity mineral oil (Shawmut Brand, Gilman Bros., Inc., Boston, Mass.). Rabbits were killed by the intravenous administration of a bolus of 40 mg/kg of sodium pentobarbital and the peritoneal cavity was lavaged four times through a #15 trochar with 50 ml portions of Dulbecco’s phosphate buffered saline containing 1: 20 (v : v) 0.1 M sodium citrate anticoagulant (final pH 6.8). Al veolar macrophages were harvested from other

38

DOHLMAN

AND

GOETZL

pentobarbital-killed rabbits by intra-tracheal lavage with ten 50 ml portions of Dulbecco’s phosphate buffered saline. Peritoneal and alveolar macrophages were recovered from the pooled lavages by centrifugation at 6009 for 15 min at room temperature and were washed twice in Medium 199 with Hanks’ balanced salt solution containing 100 units/ml of penicillin G and 100 pg/ml of streptomycin. Peritoneal cell suspensions contained over 90% macrophages and alveolar cell suspensions contained over 98% macrophages as assessed by differential counting of air-dried smears stained with Giemsa; all preparations exhibited 9570 or greater viability as assessed by trypan blue dye exclusion. Surfactant lipoprotein was prepared from the cell-free supernatant fluid of pooled tracheal lavages by centrifugation at 1OOOg for 60 min as described (11). The pelleted lipoprotein fraction from three rabbits was resuspended in 4 ml of Medium 199 buffer and stored in 0.5 ml portions at - 90°C prior to being employed in migration studies. Macrojdzage migration assay. Agarose was dissolved in boiling distilled water at a final concentration of 2 g/100 ml. The agarose solution was cooled to 55°C and mixed with an equal volume of sterile 2X Medium 199 with Hanks’ balanced salt solution that contained penicillin G (200 units/ml), streptomycin (200 pg/ml) and 0.5 g/100 ml of gelatin, and that had been pre-warmed to 48°C. Seven hundred and fifty microliters of the agarose mixture was poured into each 16 mm diameter cup in the plastic plates, allowed to harden at room temperature, and then cooled for 0.5 hr at 4°C. A pair of 3 mm diameter wells were punched through the agarose to the plastic surface in each cup with a standard spacing of 6 mm between the centers of the two holes. The agarose plugs were removed from the wells with a Pasteur pipette attached to a source of negative pressure just sufficient to lift out the plugs and leave a smooth edge. Ten microliter samplesof various concentrations of stimuli or of buffer alone were added to one well and the other received 10 ~1 of a cell suspensioncontaining 2.5 to 3.0 x lo5 peritoneal macrophages or 3.0 to 4.0 x IO5 alveolar macrophages per well in buffer alone or in a dilution of an inhibitory or enhancing factor. The plastic plates were covered and incubated in a humidified atmosphere of 5% CO* in .air at 37°C for 18 hr for peritoneal macrophages and 36 hr in the case of alveolar macrophages, to allow for maximal migration of each cell type. The plates were then flooded with absolute methanol for 15 min at room temperature and washed and stored at 4°C for 12 to 36 hr in 5% formalin buffered with sodium acetate (pH 7.1). Macrophages which migrated out of the cell well toward or away from the stimulus well were counted at a magnification of 440X. A grid positioned in the eyepiece of the microscope so as to project a width of 0.2 mm was brought up to the margin of the cell well and centered on the line connecting the two wells (Fig. 1) . Total macrophage counts in the grid extending from the margin of the cell well to the border of greatest migration were generated at two poles of each cell well, one toward and one 180” away from the stimulus well (Fig. 1) . Indices of chemokinesis and chemotaxis were calculated as indicated in Figure 1; P values were derived from a standard two sample Student’s t test. RESULTS Initial studies of the optimum conditions for the assessmentof macrophage migration under agarose indicated that the number of macrophages added to the cell well,

DETERMINANTS

OF

ALVEOLAR

MACROPHAGE

0

Buffer

ophoosr

0

Spontaneous migration

_-- A + B 2

Chewkinetic index

__-

stimulus

Chematactic index

0

Chemokinetic index

Buffer

MIGRATION

39

D A+ B/2 C-D A+ B/2

_ E+F/2 -7izE

FIG. 1. Quantitationof rabbit macrophage migrationunderagarose.The crosshatchedcircles surroundingthe macrophagewells representthe patterns of migration with buffer (1) or a stimulusin the opposingwell (Z), or with the additionof a stimulusto the macrophagewell (3). Following fixation of the agaroseplates,migration was quantitatedby microscopically countingthe numberof macrophages in 0.2 mm wide grids of length A to F which were centered on a line connectingthe two wells.

the ambient temperature, the partial pressure of CO*, and the gelatin concentration in the agarose were critical determinants. Spontaneous migration from triplicate wells was strikingly increased from a mean (* SD) of 59 f 11 with 2.5 x lo5 alveolar macrophages per well to 137 * 18 with 4.0 X lo5 alveolar macrophages per well, while the chemokinetic response to 10 ~1 of rabbit serum yielded macrophage counts of 226 f 34 and 274 1 43, respectively, for the two cell concentrations. The number of macrophagesper well was thus standardized and kept constant in any one series of experiments. A change from room temperature to 37°C gave a mean 4-fold increase in spontaneous migration and a mean 2.5-fold enhancement of C5 fragment-induced chemokinesis. Incubation of alveolar and peritoneal macrophages in a 5% COa environment stimulated spontaneousmigration and C5 fragment-induced chemokinesis from quadruplicate wells by 80 to 114% and 36 to 51%, respectively, relative to incubation in room air. Examination of the time course of spontaneous migration at 37°C in a 5% CO, environment revealed an early phase of rapid migration followed by a decrease in the rate of migration in later time intervals for both types of macrophages (Table 1). The rate of spontaneous migration of peritoneal macrophages fell after 18 hr and reached a barely perceptible level by 24 hr, while the rate of migration of alveolar macrophages achieved a peak between 24 and 36 hr and subsequently slowly declined. The chemokinetic effect of C5 fragments added to the stimulus well or the macrophage well was detectable by 6 hr for peritoneal macrophages and by 12 hr for alveolar macrophages and remained in evidence throughout the 42 hr of incubation, The chemokinetic stimulation appeared to be greater for both types of macrophages when the C5 fragments were added to the macrophage well. In all circumstances, the chemokinetic index decreased with the time of incubation and reached a plateau value by 18 to 24 hr for peritoneal macrophages and 30 to 36 hr for alveolar macrophages. The effects of the gelatin concentration in the agarose riledium on macrophage spontaneous and stimulated migration were analyzed (Fig.

40

DOHLMAN

GOETZL

AND

TABLE Time

Course

of Alveolar Migration

1

and Peritoneal Macrophage and Chemokinetic Response Time of incubation

6

12

1X

37 * 1s

112 f 26

269 f 61

1.94 2.29

1.65 1.82

1.38 1.66

24

A. Peritoneal Spontaneous migration’ Chemokinesis2 C5 fr in stimulus well C5 fr in macrophage well

12

103

1.32 1.57

0 -

1s* 2.06 2.54

7

26 f 10 1.98 2.39

312 * 94

319 f

1.34 1.51

24 B. Alveolar

Spontaneous migration’ Chemokinesis2 C5 fr in stimulus well C.S fr in macrophage well

36

42

macrophages

306 f

18

(hr) 30

Time of incubation 6

Spontaneous

125

327 f

1.30 1.46

1.39 1 so

36

42

108

(hr) 30

macrophages

62f

13

1.79 2.18

127 f 21 1.61 2.04

182 f 1.50 1.96

28

221 f

3Y

1.52 2.01

1 Spontaneous migration represents the mean f SD for three experiments utilizing 2.8 X 105 peritoneal macrophages per well and 3.6 X lo5 alveolar macrophages per well; the calculation of results is according to Fig. l-(l). 2 Chemokinesis is the response to the addition to the stimulus welt or macrophage well of C5 fragments at a concentration of 25 pg C5 equivalent per ml; the mean chemokinetic index for the three experiments is calculated according to Fig. l-_(Z) and (3). - The chemokinetic index could not be calculated.

2). Spontaneous migration increased progressively with each increment in gelatin concentration so that the level achieved at .a gelatin concentration of 0.75 g/100 ml was more than lo-fold higher than in the absence of gelatin. The magnitude of the chemokinetic response evoked by the C.5 fragments added to the macrophage well declined with increasing gelatin concentration. Increases in spontaneous migration contributed substantially to the reduction in the calculated values of the chemokinetic index [Fig. l-(3) 1. The chemotactic responses to C5 fragments in the stimulus well ranged from 6 to 38% of spontaneous migration [Fig. l-(2) ] and

400

360 320 260 240 200 160 120 60 40 0

Gelatm Concentrationan Agamse @/lOOmI)

FIG.2. Relationship of the concentration of gelatin in the agarose to alveolar macrophage migration. Results depicted are the mean of values from two agarose cups for chemotaxis and chemokinesis and the mean -t- SD of values from three cups for spontaneous migration. Each macrophage well contained 4.0 X 10” cells. C5 fragments, the stimulus employed, were prepared by tryptic digestion of 50 gg of C5 per ml and were added without dilution to the macrophage well at the initiation of the 36 hr incubation period. Chemokinesis was calculated accqrding to Fig.

l-(3).

DETERMINANTS

OF

Alveolar

ALVEOLAR

Mocrophoges

Peritoneal 0

-1.2’

Rabbit Serum

MACROPIIAGE

C5fr

MIGRATION

41

Macrophages

Chemoklnesir

F-Met Phe Leu

R&bit Serum

Csfr

F-Met Phe Leu

FIG. 3. Chemokinetic and chemotactic stimulation of macrophage migration. The assessment of alveolar macrophage migration (left-hand frame) employed 3.2 X lo5 cells per well while studies of peritoneal macrophages (right-hand frame) utilized 2.5 X lo” cells per well. Each bar represents the mean * range for three experiments in which the range of spontaneous migration of alveolar macrophages was 125 to 216 cells in 36 hr and of peritoneal macrophages was 241 to 356 cells in 18 hr. The left-hand open bar of each pair illustrating the chemokinetic responses to a given stimulus depicts the effect of adding the stimulus to the macrophage well [Fig. l-(3) 1, while the right-hand open bar shows the response obtained with the stimulus in the opposite well [Fig. l-(2) 1. Rabbit serum was used undiluted, F-Met-Phe-Leu was at a concentration of 5 X lo-* M, and C5 fragments (C5 fr) prepared by tryptic digestion of 50 pg of CS per ml were added to the wells without dilution. * P < 0.01; t P < 0.05.

were consistently less than the corresponding chemokinetic responses. In all subsequent experiments, alveolar macrophages and peritoneal macrophages were enumerated after incubation periods of 36 and 18 hr, respectively. The concentration of gelatin was set at 0.25 g/100 ml to provide readily quantitated levels of spontaneous migration and chemokinesisand to maximize the apparently minor chemotactic effects of the C5 fragments and other stimuli. As principles which chemotactically stimulate PMN leukocytes appeared to exert a predominantly chemokinetic effect on macrophage migration under agarose, the relative effects of a range of stimuli on alveolar and peritoneal macrophage chemotaxis and chemokinesis were assessed(Fig. 3). Both populations of macrophages were significantly chemokinetically stimulated by rabbit serum, C5 fragments, and synthetic F-Met-Phe-Leu, and in each casethe extent of stimulation was more significant when the factor was added to the macrophage well than when it was present in the stimulus well [Fig. l-(2), (3), and Fig. 31. Chemotaxis of alveolar macrophages was only minimally stimulated by the optimal chemokinetic concentrations of any of the factors, as the mean level of chemotactic migration was consistently less than 20% of spontaneous migration (Fig. 3). In contrast, chemotaxis of peritoneal macrophages was stimulated to moderate levels of 35 to SOY0of spontaneousmigration by all three factors. Differences in the two methods employed to assesschemokinesis (Fig. 1) were further explored by examining the dose-responserelationships for C5 fragments and F-Met-Phe-Leu in each protocol. For alveolar macrophages, maximal stimulation of the chemokinetic response was achieved with lower concentrations when

42

DOHLMAN

F-Met

AND

Phe Leu (M) Cl

q

GOETZL

Csfr $9 equivalent /ml) Stnulus in Stimulus Well Stlmulusin Macrophage Well

24 22 20 1

18

2 0 ‘$

‘6 14 I2

5

IO

i$

08

5

06 04 02

-

0 rr

-

_ 10-6 103

Concentration

1.5

3.0

6 3 125

25

50

of Stimulus

FIG. 4. Dose-response relationships of chemokinetic stimulation of alveolar macrophage migration. Each bar represents the mean results of two experiments in which the specified concentrations of F-Met-Phe-Leu or C5 tryptic fragments (C5 fr) were added to the stimulus well (left-hand bar) or to the macrophage well (right-hand bar). The quantity of alveolar macrophages in the two experiments was 3.2 X 10” and 3.4 X lo5 per well, respectively, which provided a spontaneous migration of 185 2 17 macrophages (mean k SD) in 36 hr.

factor was added to the macrophage well than when present in the stimulus well (Fig. 4). With the former protocol [Fig. l-(3) 1, peak chemokinesis was seen at 3 pg C5 equivalent/ml and 10mgto IO-* M F-Met-Phe-Leu, while with the latter protocol [Fig. l-(Z) 1, the peak of chemokinetic stimulation occurred at concentrations of 12.5 to 50 pg C5 equivalent/ml and lo-* to 1O-7 M F-Met-Phe-Leu. With peritoneal macrophages (Fig. S), differences in chemokinetic stimulation in

either

F-Met

Phe Leu (MI Cl

q

C5fr Qg equivalent/ml)

Stimulus in Stimulus Well Stimulusin Macraphage Well

2.4 2.2 2.0

9

1077 10-6 10-5 Concentration

I.5

3.0

63

125

25

50

of Stimulus

FIG. 5. Dose-response relationships of chemokinetic stimulation of peritoneal macrophage migration. Each bar represents the mean results of two experiments in which the specified concentrations of F-Met-Phe-Leu or C5 tryptic fragments (C5 fr) were added to the stimulus or macrophage well as in Fig. 4. The quantity of peritoneal macrophages in the two experiments was 2.5 X 10’ and 3.0 X 10’ per well, respectively, which provided a spontaneous migration of 336 2 24 macrophages (mean f SD) in 18 hr.

DETERMINANTS

OF

ALVEOLAR

MACROPHAGE

TABLE Influence

of Time

F-Met-Phe-Leu concentration (Ml

of Addition Responses Time

10-9

5 x

10-s

5 x

10-T

5 x

10-e

on Chemokinetic Macrophages

of F-Met-Phe-Leu

0 Chemokinesis*

5 x

2

of Stimulus of Alveolar

of addition

and Chemotactic

to stimulus

24 Chemotaxis2

2.04 2.10 1.60 1.28

43

MIGRATION

Chemokinesis

0.03 0.29 -0.10

-0.14

1 Ten microliters of F-Met-Phe-Leu solution was times and the alveolar macrophage responses were 2 Chemokinesis and chemotaxis were calculated as tion (A + B/2) was 191 f 24 macrophages (mean

1.06

0.94 0.91 0.72

well

(hr)’

0 and 24 Chemotaxis

Chemokinesis

Chemotaxis

0.03 0.28 0.24 0.09

1.77 1.84 1.51 1.27

-0.08 0.32 -0.09 -0.17

added to the stimulus wells at the specified quantitated after incubation for 36 hr. noted in Eq. (2) of Fig. 1; spontaneous migraf SD).

the two protocols were not apparent as the peak effects were seen at comparable concentrations of 6.3 pg C5 equivalent/ml and lo-* to 1O-7 Al F-Met-Phe-Leu. In order to rule out the possibility that the prolonged duration of migration under agarose favored chemokinesis over chemotaxis .as a result of dissipation of the concentration gradient of the stimulus, F-Met-Phe-Leu at varying concentrations

I

tlTf

A

I

TI f

I 106

P L lb

i

A -----i--L 104 Concentration

60

105 Id4 of Inhibitor

(Ml

FIG. 6. Effects of metabolic inhibitors on rabbit macrophage migration. Macrophages were preincubated for 30 min at room temperature with varying concentrations of sodium iodoacetate (left-hand frame) or 2,4-dinitrophenol (right-hand frame) prior to being loaded into the cell wells. The mean values (2 SD) representing 100% for alveolar macrophages were 138 (? 21) for spontaneous migration and 364 (* 69) for chemokinesis stimulated by C5 fragments at a concentration equivalent to 50 pg of C5 per ml. The mean values (2 SD) representing 100% for peritoneal macrophages were 163 (2 27) f or spontaneous migration and 253 (-t- 47) for chemokinesis stimulated by F-Met-Phe-Leu at a concentration of 1Om8 M. Suppression of migration achieved a significance level of < 0.01 for alveolar macrophage spontaneous migration with iodoacetate at lo-’ and lo-’ M and 2,4-dinitrophenol at lo-” and lO+ M, for alveolar macrophage chemokinesis with iodoacetate at lo-’ M and 2,4-dinitrophenol at 10m3 M, and for peritoneal macrophage spontaneous migration and chemokinesis with iodoacetate at IO’ M.

44

DOHLMAN

AND

GOETZL

17 Alveolar macrophoges

Surfactant FIG. 7. for three tion were sponding centrations.

I:2 I:4 Lipcqfotem

I:8 I:16 (Dilution)

&j

Peritoneal

I:32

I:64

Iwcrophcqes

Buffer Control Dipalmitoyl

6 I6 32 64 126 256 Phosplmtidylcholina @/ml)

Influence of surfactant on macrophage migration. Each bar represents the mean * SD agarose cups. In one experiment varying dilutions of a surfactant lipoprotein preparaadded to the macrophage wells and in the other experiment, shown with its correbuffer control, synthetic dipalmitoyl phosphatidylcholine was added at defined con*P < 0.01, t P < 0.05.

was added to the stimulus well at 0, 24, or both 0 and 24 hr of a 36 hr incubation interval (Table 2). Chemokinetic stimulation was dependent on the presence of the peptide in the first 24 hr as its addition at 24 hr alone failed to achieve any stimulation and a second addition at 24 hr did not influence the overall effect. Chemotaxis was only minimally stimulated and this effect was independent of the time of addition of F-Met-Phe-Leu. The effects on alveolar and peritoneal macrophage migration of the metabolic inhibitors iodoacetate, which blocks glycolysis, and 2,4-dinitrophenol, which blocks oxidative respiration, were assessed in three consecutive experiments with cells from different rabbits. Iodoacetate inhibited the spontaneous migration and chemokinesis of both populations of macrophages in a dose-related fashion with more profound suppression of the alveolar macrophage functions (Fig. 6). In contrast, 2,4-dinitrophenol inhibited alveolar macrophage spontaneous migration and chemokinesis without influencing either function of peritoneal macrophages. In view of the apparent differences between the two macrophage populations with respect both to the metabolic requirements for migration and to the capacity to exhibit a chemotactic response, an experiment was designed to study the effects on migration of surfactant, a lipoprotein product present on the mucous membranes of the respiratory tract. The addition of a crude surfactant preparation to the macrophage wells significantly stimulated the migration of alveolar and peritoneal macrophages in a dose-related fashion (Fig. 7). The extent of stimulation was comparable for the two populations of macrophages. Surfactant only slightly enhanced the chemokinetic responses of macrophages to C5 fragments and F-Met-Phe-Leu and this effect was not statistically significant (not shown). Synthetic dipalmitoyl phosphatidylcholine, a lipid component of surfactant which is responsible for a substantial portion of the effects of surfactant on pulmonary mechanics, failed to influence the spontaneous migration of alveolar macrophages (Fig. 7). DISCUSSION After their generation from distinct bone marrow precursors, monocytes circulate in peripheral blood for a brief period and then attach to sinusoids in some organs and randomly enter the tissues to be transformed into macrophages (12). Macrophages perform numerous specialized functions that appear to be dependent

DETERMINANTS

OF

ALVEOLAR

MACROPHAGE

MIGRATION

45

on the tissue site in which they localize. In mammalian hmg, macrophages are involved in clearance of inhaled particulate matter, hacteriostasis, generation and degradation of chemical mediators, and activation of fibrinolytic pathways (1, 13-15). While the flexibility and inducibility of numerous macrophage constituents and functional capabilities have been demonstrated (16), less is known of the relationship of these adaptations to mlique local tissue conditions. The demonstration that alveolar and peritoneal macrophages exhibit both shared and divergent characteristics of migration has implications relevant to the tissue maturation of macrophages. A range of factors known to stimulate predominantly chemotactic behavior in polymorphonuclear leukocytes, including autologous sermii, tryptic fragments of the fifth component of complement, and the synthetic pepticle F-h4et-Phe-Leu, evoked substantial chemokinesis of both alveolar and peritoneal macrophages (Figs. 1, 3). The addition of C5 fragments to the macrophage well or the stimulus well resulted in a chemokinetic response which was detectable within 6 to 12 hr of incubation and was evident for over 42 hr (Table 1). Although peritoneal macrophages exhibited greater chemotaxis than alveolar macrophages, the magnitude of the response of either macrophage population was far lower than their corresponding chemokinetic response. Chemokinesis was the major migratory response whether the factors were added to the stimulus well or directly to the macrophages (Fig. 3) and whether the factors were introduced only at the beginning of a 36 hr migration period or initially and again after 24 hr of migration (Tahle 2). Further, chemokinetic stimulation was the predominant response over a wide range of conditions which strikingly influenced the magnitude of the migratory response including variations of gelatin concentration (Fig. 2)) ambient temperature, CO2 partial pressure, and the concentration of stimuli (Figs. 4, 5). The application of specific inhibitors distinguished the metabolic requirements for migration of alveolar and peritoneal macrophages (Fig. 6). Iodoacetate, which blocks the glycolytic metabolism of glucose, provided a close-related inhibition of spontaneous migration and chemokinesis of both types of macrophages. In contrast, 2,4dinitrophenol, which blocks oxidative metabolism, distinguished between the two macrophage populations. Alveolar macrophage migration and chemokinesis were suppressed by 2,4-dinitrophenol, whereas no effect was seen on the migration of peritoneal macrophages. The demonstration of comparable metabolic requirements for macrophage migration under agarose and for macrophage phagocytosis (3), confirms the oxidative dependence of the alveolar macrophage as compared to the peritoneal macrophage. Surfactant lipoproteins are surface n~u~~us constituents which modulate the normal mechanical behavior of the lung (17). The addition of portions of a preparation of surfactant to alveolar and peritoneal macrophages enhanced their spontaneous migration in a dose-related fashion (Fig. 7). Dipalmitoyl phosphatidylcholine, the major lipid component of surfactant (IS), only minimally stimulated alveolar macrophage migration at concentrations comparable to those in surfactant preparations (11). The stimulation of migration of alveolar macrophages by a secretory product characteristically localized in the lung provides possible further evidence for the conditioning of macrophage function by the local tissue environment. This finding is also consistent with previous data demonstrating a striking stimulation of the in vitro bactericidal activity for S. ~~UVUS of rat alveolar macrophages fol-

46

DOHLMAN

AND

GOETZL

lowing the addition of a surfactant preparation (19). Further studies of the nmlecular bases for the differences in behavior of macrophages from different organ systems will be dependent on analyses of the patterns of local tissue development of unique membrane receptors and other constituents which are critical to a variety of functions. REFERENCES 1. Goldstein, E., Lippert, W., and Warshauer, D., J. Clin. Immt. 54, 519, 1974. 2. Morrow, P. E., In “Airway Dynamics” (A, Bouhuys, Ed.), pp. 219-312. C. C Thomas, Springfield, Ill. 1970. 3. Oren, R., Farnham, A. E., Saito, K., Milofsky, E., and Karnovsky, M. L., J. Cell Biol. 17, 487, 1963. 4. Pollock, E. M., Pegram, C. N., and Vazquez, J. J., 1. Reticulomdothel. Sot. 9, 383, 1971. 5. Leu, R. W., Eddleston, A. W. L. F., Good, R. A., and Hadden, J. W., Exp. Cell Res. 76, 458, 1973. 6. Ward, P. A., J. Exp, Med. 128, 1201, 1968. 7. Tainer, J. A., Turner, S. R., and Lynn, W. S., Am. J. Pathol. 81, 401, 1975. 8. Sahu, S., and Lynn, W. S., Inj?anzwtatio~z 2, 47, 1977. 9. Nilsson, U. R., Tomar, R. H., and Taylor, F. B. Jr., Znt~~lzzllzochc~~nistryy 9, 907, 1972. 10. Goetzl, E. J., I~~z~nu~zology 29, 163, 1975. 11. Harwood, J. L., Desai, R., Hext, P., Tetley, T., and Richards, R., Biochenz. J. 151, 707, 1975. 12. Gordon, S., and Cohen, Z. A., Intern Rev. Cytol. 36, 171, 1973. 13. Fishman, A. P., and Peitra, G. G., New Eng. 1. Med. 291, 884, 1974. 14. Unkeless, J. C., Gordon, S., and Reich, E., J. Exp. Med. 139, 834, 1974. 15. Sorokin, S. P., and Brain, J. D., Amt. Rec. 181, 581, 1975. 16. Cohn Z. A., Fed. Proc. 34, 1725, 1975. 17. Clements, J. A., Neze Elzg. J. Med. 272, 1336, 1965. 18. Goerke, J., Biochk Biophys. Acta 344, 241, 1974. 19. LaForce, F. M., Kelly, W. J., and Huber, G. L., Rev. Respir. Dis. 108, 784, 1973.