Modulation of lymphocyte motility by macrophages

CELLULAR

109,295-305

IMMUNOLOGY

Modulation

(1987)

of Lymphocyte

Motility by Macrophages

H. TAK CHEUNG AND J.-S. Twu Department

ofBiological

Sciences,

Illinois

State University,

ReceivedFebruary17,1987;acceptedA4ayll,

Normal,

Illinois

61761

1987

Motility of lymphocytes plays a significant role in their functions. Because macrophages frequently associate with lymphocytes in lymphoid tissues and inflammatory sites, they are likely to be important in regulating lymphocyte motility. In this study, we identified a chemokinetic activity in macrophage culture supematants. Interestingly, this activity could be detected by the capillary migration assay but not by the more commonly used Boyden chamber chemotaxis assay.Colchicine, on the other hand, was chemokinetic for lymphocytes in the Boyden chamber chemotaxis assaybut not in the capillary migration assay. Both these observations and previous studies on the morphology of motile lymphocytes on two-dimensional (2-D) surfaces (capillary migration assay)and in 3-D matrices (Boyden chamber chemotaxis assay)suggest that lymphocytes possessmore than one motility mechanism-one for 2-D surfaces and one for 3-D matrices. We propose that the macrophage-derived chemokinetic activity described herein only affected the motility mechanism on 2-D surfaces. In addition, we also observed that the chemokinetic activity was produced by “resting” macrophages and could not be augmented by further activation. Finally, the effect was greatest on mature T cells. We propose that this factor plays an important role in facilitating cell interactions within lymphoid tissues and inflammatory Sites.

0 1987 Academic

Press, Inc.

INTRODUCTION

The motility of lymphocytes is important in their immunological functions. For example, crossing the high endothelial venules and capillary endothelia has been shown to be dependent on the motility of lymphocytes ( I-3). Motility is also required in killing target cells (4-6), and in the interactions between lymphocyte subpopulations (7, 8) and with accessory cells, such as macrophages (9-12) and reticular cells (13). Lymphocyte motility can be regulated by immune mediators and other biological agents. In vitro chemotaxis assay has identified chemotactic activity for lymphocytes in extracts of delayed hypersensitivity skin reaction sites ( 14), thymus (15), and skin granuloma ( 16). Furthermore, culture supernatants from mitogen-activated lymphocytes (17-20) and from “resting” and activated macrophages are also chemotactic for lymphocyes ( 18,2 1). These agents could play an important role in recruiting antigennonspecific lymphocytes to sites of immunological reactions. In delayed-type hypersensitivity reactions, macrophages accumulate prior to lymphocytes (22). Macrophages are also abundant in lymph node medulla, where they present antigens to lymphocytes (23). These observations suggest an important role for macrophages in regulating lymphocyte motility by influencing their accumula295 0008-8749/87$3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tion and cell-cell interactions. Evidence in support of this is that when cultured with macrophages, lymphocytes were observed to actively migrate adjacent to the surface of macrophages (24). Together, these findings suggest the production of soluble mediators that modulate lymphocyte motility. Two studies have reported such factors. An early study reported lymphocyte chemotactic activity in culture supernatants of rat macrophages ( 18) and a recent study reported the chemotactic effect of the human monokine interleukin-1 (IL-l) (2 1). In this study, we looked for a similar factor in mice. We found that mouse macrophage culture supernatants were chemokinetic for mature T cells. The effect was less on B cells. This activity, interestingly, could be detected by the capillary migration assay but not by the more commonly used Boyden chamber chemotaxis assay. Based on our results, we suggest that lymphocytes possess two motility mechanisms; only one of the mechanisms is modulated by this chemokinetic activity. METHODS

AND MATERIALS

Animals, culture medium, and chemicals. Male C57BL/lO mice, 6-8 weeks of age, were obtained from the mouse breeding colony at this institute. EHAA medium was prepared according to Peck and Bach (25) and was supplemented with 0.5% autologous mouse serum (EHAA-S) and antibiotics (penicillin, 100 units/ml, and streptomycin, 100 pg/ml, both obtained from Sigma Chemical Co., St. Louis, MO). Puromycin and indomethacin were also obtained from Sigma Chemical Co. Isolation of spleen lymphocytes. Mice were killed by vaporized ether. Spleens were aseptically removed and dispersed into single-cell suspensions by rubbing against a 100-pm stainless-steel wire mesh in Dulbecco’s phosphate-buffered saline (PBS). Red blood cells were lysed by Tris-buffered ammonium chloride solution (0.18 mol of NH4C1 in 0.0 17 M Tris-HCl, pH 7.2) for 3 min at room temperature. The cells were washed three times with PBS, collected by centrifugation, and resuspended in EHAAS. Macrophages were depleted by first incubating the cells in 75-cm2 tissue culture flasks (Costar, Cambridge, MA) at a concentration of 2.5 X lo6 cells/ml for 1 hr at 37°C in a humidified atmosphere of 5% CO, in air. The nonadherent cells (2.5 X 108> were passed through a 0.5 X IO-cm Sephadex G-10 column (Sigma Chemical Co.). Cells passing through the column were collected by centrifugation, washed, and resuspended in EHAA-S according to the concentrations given below, More than 98% of these cells were determined to be lymphocytes by staining for nonspecific esterase (26). Separation of T and B cells with a nylon-wool column. Spleen T and B cells were separated with a nylon-wool column (27). Briefly, nylon wool (Fenwall Laboratories, Deerfield, IL) was boiled in deionized water, washed with five changes of water, and dried at 37°C for 2-3 days. Two grams of dry nylon wool were packed into a 30-ml plastic syringe, which was then autoclaved, rinsed with 20 ml of EHAA containing 5% fetal calf serum (GIBCO, Grand Island, NY), and incubated at 37°C. Spleen lymphocytes, 2.5 X 10’ cells in 6 ml of EHAA, were added to the column and incubated for 45 min at 37°C. The nonadherent cells were eluted with 60 ml of prewarmed medium at the rate of 1 drop/set. Adherent cells were removed by flushing cold PBS through the column with a syringe plunger. Cells from the column were washed once with PBS and collected by centrifugation. The adherent cells were > 95% positive for surface Ig as determined by immunofluorescence using rabbit anti-mouse Ig (Miles

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Scientific, Naperville, IL), and the nonadherent cells were >99% negative for surface Ig and >95% positive for Thy 1 as determined by immunofluorescence with anti-Thy 1 antibodies (Miles Scientific). Isolation of peritoneal macrophages and preparation of culture supernatants. Peritoneal macrophages were obtained either with or without intraperitoneal injection of 0.2 ml of complete Freund’s adjuvant (CFA) (GIBCO), 0.2 ml of mineral oil, or 0.5 ml of thioglycollate medium (DIFCO, Detroit, MI). Peritoneal macrophages were isolated by incubating the peritoneal cells in 25-cm2 tissue culture flasks (Costar) at 37°C in a humidified atmosphere of 5% CO2 in air. After 2 hr, nonadherent cells were removed by washing the flasks five times with PBS. The adherent cells were treated with rabbit anti-mouse Ig (Miles Scientific) and anti-Thy 1 (Miles Scientific) in the presence of guinea pig complements (GIBCO). The adherent cells were >98% macrophages as verified by nuclear morphology and nonspecific esterase distribution (26). The macrophage culture supernatants were obtained after incubation in EHAA-S for 24 hr. In select experiments, lipopolysaccharide (Escherichia coli, 0 127:B8; DIFCO) was added to the culture at a concentration of 20 pg/ml. Cell viability was not affected at this concentration, and the macrophages were demonstrated to have acquired tumoricidal activity (28). The culture supernatants were then collected, filtered through O.Zpm-pore filters (Millipore Corp., Bedford, MA) and stored at -55°C until used. Separation of thymocyte subpopulations by peanut agglutinin (PNA). Thymocyte subpopulations were separated by the procedure described by Reisner et al. (29). Thymuses were aseptically excised from 6-week-old mice. Any connective tissue associated with the thymic capsule was dissected away to ensure removal of the parathymic lymph nodes. The thymuses were dispersed into single-cell suspension by rubbing against a 100~pm stainless-steel wire mesh in PBS. Cells were washed twice in PBS, resuspended in PBS to 8 X lo8 cells/ml, mixed with an equal volume of PNA (Sigma Chemical Co.) (1 mg/ml in PBS), and incubated for 10 min at 22°C. To separate the agglutinated cells from the nonagglutinated cells, the cell suspension was gently layered on top of 10 ml of PBS containing 2% bovine serum albumin (Sigma Chemical Co.). After 30 min, the nonagglutinated cells were recovered by removing the top layer. The agglutinated cells settled in the bottom layer and were recovered by incubating with 15 ml of 0.2 M D-galactose at 22°C for 5 min. Cells were collected by centrifugation and washed once with PBS. Isolation of cortisone-resistant thymocytes. Mice were injected with 2 mg of hydrocortisone acetate (Merck Sharp and Dohme, West Point, PA) intraperitoneally. Forty-eight hours later, thymuses were removed, and thymocytes were prepared as described above. Cell recovery was lo- 12% of that of control mice injected with saline. Chemotaxis assay. Lymphocyte motility was determined by the modified Boyden chamber chemotaxis assay (30) using blind-well chemotaxis chambers (Nuclepore Corp., Pleasanton, CA). A lymphocyte suspension was adjusted to a concentration of 2.5 X 10’ cells/ml in EHAA-S. EHAA-S or various dilutions of macrophage culture supernatants (0.2 ml) were placed in the lower chamber, upper chamber, or both chambers; cell suspension (0.2 ml) was placed in the upper chamber. Either a Nuclepore filter (5 pm pore size) or a Millipore nitrocellulose filter (8 pm pore size) was placed between the lower and upper chambers. The chambers were incubated for 3 hr at 37°C in a humidified atmosphere of 5% CO2 in air. For the Nuclepore filters, cell motility was assessed by determining the number of cells in the lower chamber with a hemocytometer. For the nitrocellulose filters, the filters were fixed and stained

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TABLE 1 Checkerboard Analysis of Macrophage Supematants on Lymphocyte Migration Using Boyden Chambers with Nitrocellulose Filters Percentage supematant concentration above filter

Percentage supematant concentration below filter

0

6.25

12.50

25

0

loo”

101 t 2.3’ (99 k 7.6)” 100 f 4.5 (107+7.2)

104 r 3.2 (103 + 9.9)

101 + 5.3 (109 f 4.6)

6.25 12.50 25 50 75

98 f 4.8 (100&8.3) 11027.8 (102 f 4.9) 95 5 5.3 (96 + 9.5) 95 +9.1 (105 + 4.9) 105 + 8.0

50

15

49 + 4.8 (102 + 5.6) 93 2 10.2

107 + 8.4 (94 r 8.1) 103 f 4.8 (95 + 8.8) 99 + 5.2 (107k7.1) 96 zk 5.0

a Raw data in control experiment was 10.44 + 2.2 cells/HPF. This and the experiments presented in the subsequent tables were performed in quadruplicate. b Lymphocyte migration into nitrocellulose filters in the presence of supematants of macrophages cultured in medium was expressed as a percentage of control migration + SD. c Same as above except in the presence of supematants of macrophages cultured in medium with 20 r.& ml of LPS.

with hematoxylin. The numbers of lymphocytes which migrated to a distance of 5060 pm from the top of the filter in five random fields under a magnification of 400 were determined. The data were expressed as the number of cells per high-power field (HPF). All samples were tested in quadruplicate chambers. Capillary migration assay. The capillary migration assay was performed as described by Cheung et al. (31). Approximately 4 X lo6 lymphocytes in 0.07 ml of EHAA-S were drawn into sterile 15 X loo-mm glass capillary tubes and sealed at one end with 60:40 mixture of paraffin and Vaseline. The capillaries were centrifuged at 12Og for 6 min and cut at the cell-fluid interface. Two stubs were placed in each well of a migration plate (Sterilin, Teddington, Middlesex TW 11802, England). The wells were filled with approximately 0.6 ml of EHAA-S or macrophage culture supematant and incubated for 24 hr at 37°C in a humidified atmosphere of 5% CO2 in air. The migration area was projected onto a piece of paper using a microprojector and measured with a planimeter. Results were expressed as arbitrary units of migration area. All experiments were performed in quadruplicate. RESULTS Peritoneal macrophages were extensively purified as described above to ensure less than 2% contamination of other cell types. Furthermore, to avoid activation, they were cultured in medium supplemented with 0.5% autologous serum for 24 hr before removing the supematants for experiments, Lymphocytes were also rigorously purified to remove adherent cells, to achieve a purity of 98% or greater. The blind-well chemotaxis chambers were used with either nitrocellulose or Nucleopore filters. Various concentrations of supematants were placed in the upper and lower chambers according to the checkerboard analysis (32). Table 1 presents

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TABLE 2 Checkerboard Analysis of Macrophage Supematants on Lymphocyte Migration Using Boyden Chambers with Nuclepore Filters Percentage supematant concentration below filter

0

6.25

12.50

25

50

75

0

100”

11124.76 (97 + 8.5)’ 96 f 6.8 (93 + 7.2)

94 + 5.6 (102 + 3.9)

99 + 8.7 (109 f 8.1)

98 + 5.8 (95 + 8.9)

103 + 8.2

6.25 12.50 25 50 15

Percentage supematant concentration above filter

105 + 3.3 (107 f 8.8) 94 + 5.8 (105 f 5.9) 95 f 3.0 (93 + 9.1) 93 + 6.2 (103k4.9) 95 + 5.6

99+6.1 (98 f 7.2) 103 f 4.4 (107 t 5.1) 102 f 2.2 (108 f 8.3) 94 f 4.3

’ Raw data in control experiment was 1.39 f 0.03 X 10“ cells. b Lymphocyte migration into Nuclepore filters in the presence of supematants of macrophages cultured in medium was expressed as a percentage of control migration + SD. c Same as above except in the presence of supematants of macrophages cultured in medium with 20 &ml of LPS.

the results of lymphocyte migration determined with nitrocellulose filters. The checkerboard analysis revealed neither chemotactic nor chemokinetic activity in the culture supematants. Identical results were obtained with Nuclepore filters (Table 2). The capillary migration assay measures the migration of lymphocytes from capillary tubes onto tissue culture plastics (33). The area covered by migrating cells is a determination of their motility. When this assay was used to determine the effect of macrophage supernatants on the migration of lymphocytes, the migration was 309 and 225% of control at supematant concentrations of 50 and 25’46, respectively (Fig. 1). The supematants from macrophages cultured in medium or in medium contain-

0

20 SIJPERNATANT

FIG. 1. Spleen lymphocyte motility by capillary migration assay.Lymphocyte migration in the presence of culture supematants from macrophages cultured with either medium (0) or medium containing LPS (0) (20 &ml). Control migration was 3.58 + 0.3 1 units of migration area.

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FIG. 2. Spleen lymphocyte motility as determined by the capillary migration assay in the presence of culture medium (A), 50% culture supematant from resident macrophages (B), and 50% culture supematant from resident macrophages treated with LPS (C).

ing 20 pg/ml of LPS had the same effect on migration of lymphocytes (Fig. 1). Figure 2 illustrates the migration of lymphocytes with and without macrophage supematants in the medium. To make sure that the Boyden chamber chemotaxis assay was working, we tested the effect of colchicine on migration of lymphocytes. Colchicine is a lymphocyte chemokinetic agent; its effect can be detected by the Boyden chamber chemotaxis assay using either nitrocellulose (34) or Nuclepore filters. As shown in Table 3, at

TABLE 3 Effect of Colchicine on Lymphocyte Migration as Determined by Boyden Chamber Chemotaxis Assay and Capillary Migration Assay Boyden chamber chemotaxis assay

Medium (Control) Colchicine lo-“ M lo+ M lO-6 M IO-‘M IO-* M lO-9 M lo-“M

Nitrocellulose filters

Nuclepore filters

Capillary migration assay

100”

1OOb

100’

85 f 134* 225 + 288rt 125 + 114f 93+

3.8 4.1 10.2 9.8 4.9 7.7 8.1

71 + 154 f 241 ? 295 t 130 + 125 + 105 +

2.7 4.6 5.6 8.8 3.4 8.5 2.8

LIRaw data in control experiment was 12.7 -+ 1.8 cells/HPF. b Raw data in control experiment was 1.02 + 0.11 X 1O4cells. c Raw data in control experiment was 3.56 k 0.21 units of migration area.

91 zk 3.4 95 f 5.1 121 f 8.9 109 rt 7.9 118 -t 7.2 99 + 2.6 102 -t 6.5

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TABLE 4 Activation of Macrophages and the Effect of Their Culture Supematants on Lymphocyte Motility Capillary migration assay Percentage concentration of culture supematant tested Medium Culture supematants Resident PE macrophages CFA-induced PE macrophages Mineral oil-induced PE macrophages Thioglycollate-induced PE macrophages

15 25 15 25 75 25 15 25

Macrophages cultured in medium

Macrophages cultured in the presence of LPS

Migration area + SD

Percentage of control migration

Migration area + SD

Percentage of control migration

3.24 + 0.33“

100

3.02 f 0.45 b

93

9.82 f 0.57 6.7 I f 0.23 9.57 + 0.49 6.93kO.15 10.42 f 1.02 7.12eO.63 9.78 f 0.66 6.87 f 0.27

303 207 295 214 322 220 302 212

a Migration of lymphocytes in medium serving as control. b Migration of lymphocytes in medium containing 20 &ml

10.03 7.01 9.84 6.98 9.42 6.73 9.66 6.66

f 0.67 f 0.46 * 0.93 f 0.6 1 f 0.78 f 0.45 + 0.36 -+ 0.88

310 216 304 215 291 208 298 206

of LPS.

the optimal concentration (lo-’ M), migration was 288 and 295% of control for the nitrocellulose and Nuclepore filters, respectively. In the capillary migration assay, interestingly, colchicine was not chemokinetic for lymphocytes in the concentrations tested (Table 3). Thus, in different assay systems, the chemokinetic responses of lymphocytes appear to be different. In an attempt to augment the chemokinetic activity in macrophage culture supernatants, mice were injected intraperitoneally with inducing agents (CFA, mineral oil, or thioglycollate) before isolating the peritoneal macrophages. The macrophages were cultured in medium with or without LPS for 24 hr before collecting the supematants. As shown in Table 4, no difference in chemokinetic activity was observed in supematants from the resident and induced macrophages. Furthermore, the presence of LPS (20 pg/ml) during culture did not increase the chemokinetic activity. The effect of indomethacin (an inhibitor of cyclooxygenase) and puromycin (an inhibitor of protein synthesis) on the production of chemokinetic activity was determined. The compounds were added to the medium during culture. Indomethacin had no effect, whereas puromycin inhibited the production of chemokinetic activity (Table 5). Neither indomethacin nor puromycin alone had any effects on the migration of lymphocytes, as indicated in Table 5. To determine the cell types that responded to the macrophage supematants, spleen lymphocytes were separated into T and B cells by a nylon-wool column. Immunofluorescence revealed less than 5% contamination by the other cell type. In the presence of medium, T cells were twice as motile as B cells. In the presence of macrophage supematants, the migration of T and B cells was 290 and 166% of the respective controls (Table 6). Thymocytes, however, did not respond chemokinetically to the macrophage supematants. To determine whether only mature T cells respond to the

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TABLE 5 Treatment of Macrophage Culture with Indomethacin and Puromycin and the Effect of Their Supematants on Lymphocyte Migration Capillary migration assay

Treatment

Migration area in the presence of medium + reagents

Migration area in the presence of macrophage supematant

Percentage increase in migration”

Medium (control) Indometbacin (4 X IO-‘M) Puromycin (1 fig/ml)

3.84 + 0. I 1’ 3.34 k 0.08 4.11 kO.21

11.21 f0.18 10.92 20.18 5.225 1.11

292 327 127

a Percentage increase in migration in the presence of macrophage supematants as compared to that in medium containing the same reagents. b Migration area + SD (units).

chemokinetic factor, mature thymocytes were obtained by agglutination with PNA or by injecting mice with hydrocortisone. As shown in Table 6, mature thymocytes (PNA-nonagglutinated and cortisone-resistant) isolated by both procedures responded chemokinetically to the macrophage supematants, whereas immature thymocytes (PNA-agglutinated) did not respond. DISCUSSION We believe that we have identified a unique macrophage-derived mediator that modulates the motility of lymphocytes. This mediator has an interesting property: it TABLE 6 Migration of Different Lymphocyte Subpopulations in the Presence of Macrophage Culture Supematants Capillary migration assay

Lymphocyte subpopulations Spleen lymphocytes Unseparated Spleen T cells Spleen B cells Thymocytes Unseparated PNA-agglutinated cells PNA-nonagglutinated cells Cortisone-resistant thymocytes

Migration area in the presence of culture medium

Migration area in the presence of macrophage supematant

Percentage increase in migration”

3.40 + 0.28b 4.27 k 0.48 1.81 kO.09

9.92 f 0.18 12.37 f 1.11 3.01 -c 0.26

292 290 166

2.22 2 0.04 1.78 k0.31 3.27 + 0.26 3.09 +- 0.25

2.43 r 0.12 1.93 -t 0.09 8.33 -c 0.54 8.91 fO.11

109 108 255 288

u Percentage increase in migration in the presence of macrophage supematants as compared to that in medium. b Migration area + SD (area).

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is chemokinetic for lymphocytes in the capillary migration assay, but it has no effect in the Boyden chamber chemotaxis assay. Several possible explanations for this phenomenon can be put forward. The most likely explanation is that lymphocytes possess different motility mechanisms that are measurable by different assays. The macrophage factor modulates one of the mechanisms; therefore, the effect is detected in only one of the assays. Several lines of evidence led to this conclusion. First is the past observations of lymphocyte motility on different types of substratum. The motility of lymphocytes was first reported by McCutcheon (35) and was later extensively studied by Lewis (36, 37). In these studies, lymphocytes migrated from plasma clot onto two-dimensional (2-D) plane glass surfaces. The motile lymphocyte has a unique morphology which is quite different from that of fibroblasts and macrophages. It comprises a round cell, which contains the nucleus, in the forward direction, and a trailing cytoplasmic tail (36-38). On the other hand, motile fibroblast is polygonal in shape (39, 40). Lymphocytes form weak and limited adhesive contacts to the substratum (35), whereas fibroblasts form tight and multiple contacts (39). Finally, the motility rate of lymphocytes (41) under this condition is much higher than the motility rate of fibroblasts (39). But, when lymphocytes migrate within 3-D matrices, such as hydrated collagen gel and latticed filters, their morphology resembles that of fibroblasts, with pseudopodia extending in different directions (42). Therefore, the morphological differences of lymphocytes migrating in different environments indicate that perhaps lymphocytes possess more than one type of motility mechanism. The second evidence is the effect of colchicine on lymphocyte motility. Colchicine has a chemokinetic effect on lymphocytes, presumably by disrupting the integrity of the microtubules (34). In this study, colchicine was, however, only chemokinetic in the Boyden chamber chemotaxis assay; it had no effect in the capillary migration assay. To explain this discrepancy, the differences between these two assays should be discerned. Although both methods assay cell motility, there are many differences between the two systems. But, none is greater than the difference in the substratum. In the capillary migration assay, lymphocytes migrate from capillary tubes onto a 2D open surface, a substratum similar to that described in Lewis’ studies. On the other hand, lymphocytes migrate into filters, 3-D lattices, in the Boyden chamber chemotaxis assay. And so, colchicine apparently only affects migration into a 3-D lattice and has no effect on the motility on a 2-D open surface. Based on this evidence, it is reasonable to conclude that the macrophage-derived chemotactic activity also has a selective but opposite effect on lymphocytes: it affects their motility on a 2-D surface, therefore detectable in the capillary migration assay, but has no effect on their migration into a 3-D lattice. We also found that this macrophage chemokinetic activity differs in several respects from the macrophage-derived chemotactic factor reported by Ward et al. ( 18) and IL- 1 reported by Mossec et al. (2 1). First, Ward’s factor was produced by resting macrophages but could be augmented by activation ( 18). IL- 1 is produced only by activated macrophages (2 1). In this study, however, we did not observe any difference in the production of the chemokinetic activity between resting and activated macrophages. Second, the effect of Ward’s factor and IL-l was detectable in the Boyden chamber chemotaxis assay and was mainly chemotactic (18, 2 1), whereas we could only detect the activity in the capillary migration assay. Lastly, Ward’s factor (18) was similar to ours in its effect on subpopulations: a greater effect on T cells than on

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B cells. But, the effect of IL-l is contrary: a greater effect on B cells than on T cells (21).

Several possibilities could account for these differences. First is species difference: Ward’s study used rats (18), Mossec et al. used human IL- 1 and mononuclear cells (2 l), and we used mice. Second are the differences in experimental conditions. This can be resolved by using their materials in our system and vice versa. Such arrangements will be made. Third are the sources of macrophages. Ward’s and our study used peritoneal macrophages (18), whereas Mossec et al. used peripheral blood monocytes (2 1). As a result, macrophages from different sources could produce factors with distinct properties. Finally, in support of the uniqueness of this factor, we showed that it is not a cycloxygenase metabolite; a previous study has demonstrated lymphocyte chemokinetic activities of such metabolites (43). The exact nature of this factor is still unclear, however, and purification and biochemical characterization is currently underway. What is the role of this macrophage-derived chemokinetic activity? We predict that its primary role is to facilitate cell interactions. When antigen-nonspecific lymphocytes enter inflammatory sites and lymph node medulla, they interact with accessory cells, such as macrophages as well as other cells. Studies by scanning electron microscopy revealed that both inflamed tissues and lymph node medulla are likely to resemble a 2-D surface (44). In these sites, this chemokinetic activity produced by macrophages or other accessory cells enhances lymphocyte motility and subsequently facilitates cell interactions. Because the lymphocytes are not induced to migrate into 3-D matrices of the surrounding undamaged tissues, they will be confined in this area. Our data suggest this to be a reasonable hypothesis. ACKNOWLEDGMENTS This work was supported by Grant AI 20343 from the National Institutes of Health (Bethesda, MD) and by an Organized Research Grant from Illinois State University. The authors thank Ms. Joni St. John for assisting in the preparation of the manuscript.

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