Vol. 21, No. 1

INFECTION AND IMMUNITY, July 1978, p. 254-268 0019-9567/78/0021-0254$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Murine Macrophage-Lymphocyte Interactions: Scanning Electron Microscopic Study R. M. ALBRECHT,`* R. D. HINSDILL,2 P. L. SANDOK,' AND S. D. HOROWITZ2 Department of Bacteriology' and Department of Pediatrics, Division of Immunology,' University of Wisconsin, Madison, Wisconsin 53706 Received for publication 21 December 1978

Light and scanning electron microscopic observations revealed murine macrophage-lymphocyte interactions involving the initial contact of peritoneal, spleen, or thymus lymphocytes with peritoneal macrophage processes or microprocesses followed by clustering of lymphocytes over the central nuclear area of the macrophages. Lymphocyte-lymphocyte clustering was not observed in the absence of macrophages. Attachment and subsequent clustering appeared not to require the presence of serum or antigen; the attachment of allogeneic or xenogeneic lymphocytes was comparable to that seen in the syngeneic system, but central clustering of these lymphocytes failed to occur. No attachment or clustering was observed when thymic lymphocytes were cultured with thymus-derived fibroblasts rather than with peritoneal macrophages. Lymphocyte attachment to immune, antigen-activated, syngeneic macrophages occurred more rapidly than that to normal unstimulated syngeneic macrophages; however, lymphocytes attached to the "activated" macrophages appeared to be killed by a nonphagocytic mechanism. A similar increase in the rate of lymphocyte attachment to macrophages occurred in the presence of migration inhibitory factor. Subsequent lymphocyte clustering on macrophages was observed in the migration inhibitory factor-stimulated cultures. In addition, lymphocyte-macrophage interactions similar to those in vitro were observed to occur in vivo on intraperitoneally implanted cover slips. The interaction of lymphocytes with or in the presence of macrophages has been shown to be important in a number of immune responses, e.g., primary and secondary antibody responses to T-dependent as well as certain T-independent antigens (4, 8, 15, 41, 66), T-cell antigen recognition and T-helper-cell generation (11, 25, 53), certain lymphocyte blastogenic responses (7, 50, 60, 68), syngeneic tumor immunity (13, 49, 69), and in mixed leukocyte culture reactivity (52). Macrophage-lymphocyte interactions have been shown to occur both in vivo (38, 59) and in vitro (20, 55, 62-64, 68; R. Albrecht, R. Hinsdill, P. Sandok, A. MacKenzie, and I. Sachs, Program Abstr. 9th Leukocyte Culture Conf., Williamsburg, Va., 1974, Abstr. no. 6), and, although they appear not to require the presence of antigen or serum (14, 31, 32; Albrecht et al., Program Abstr. 9th Leukocyte Culture Conf., 1974, Abstr. no. 6), these interactions may be modified in the presence of such factors (35, 36, 53; Albrecht et al., Program Abstr. 9th Leukocyte Culture Conf., 1974, Abstr. no. 6). The present study was undertaken to determine the nature of the direct lymphocyte-macrophage interaction with respect to the physical

orientation and surface morphology of the interacting cells. MATERIALS AND METHODS Animals. Ten- to 20-week-old female inbred BALB/c or Swiss-Webster ROR outbred mice were used as the source of murine cell populations. For studies utilizing neonatal cells, 8- to 12-h-old BALB/c mice were used. Human thymic tissue was obtained at the time of corrective heart surgery. Media. Medium NCTC-109 (Microbiological Associates, Bethesda, Md.) with L-glutamine and 50 ,tg of gentamicin per ml was used for all cell cultures. Where noted, this was supplemented with fetal calf serum (International Scientific Industries, Cary, Ill.) at the concentrations listed. Cells were washed in Hanks balanced salt solution (Grand Island Biological Co., Grand Island, N.Y.). Macrophages. Normal, unstimulated murine peritoneal macrophages were obtained by the injection of 2 ml of medium NCTC-109 into the peritoneal cavity. After brief agitation, the fluid was removed and found to contain approximately 5 x 106 cells per ml (20 to 30% macrophages, 70 to 80% lymphocytes). Approximately 0.05 to 0.075 ml of the suspension was deposited on 12-mm-diameter, round glass cover slips (Owens Illinois, Vineland, N.J.) previously washed and sterilized. The cover slips were placed in small plastic petri dishes (Falcon no. 3001), and the suspended cells were

254

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

allowed to settle out and adhere for 7 to 10 min at room temperature. Nonadherent cells were then washed free with a stream of Hanks balanced salt solution. Adherent cells showed greater than 95% viability as assessed by trypan blue exclusion and contained less than 2% residual lymphocytes. Lymphocytes. Splenic and thymic lymphocytes were obtained by dispersion of the respective organs through a fine nylon mesh, followed by suspension of the dispersed cells in Hanks balanced salt solution. Cells were then separated on a Ficoll-Hypaque gradient and washed three times in Hanks balanced salt solution. The resultant cell populations were greater than 95% viable by trypan blue exclusion. Cells were adjusted to a concentration of 107/ml in NCTC-109. Human thymic lymphocytes were prepared in a similar manner. Fibroblast monolayers. Fibroblast monolayers were obtained by culturing small pieces of minced thymus on glass cover slips in medium NCTC-109 at 370C in a 5% C02-95% air atmosphere. Well-developed monolayers were present by 7 to 10 days of culture. In vitro lymphocyte-macrophage cultures. In initial studies, peritoneal lymphocytes were allowed to settle among adherent autologous macrophages on cover slips (2; Albrecht et al., Program Abstr. 9th Leukocyte Culture Conf., 1974, Abstr. no. 6). Subsequent studies used syngeneic peritoneal lymphocytes or splenic or thymic lymphocytes. These cells were added to macrophage cultures to produce a final ratio of five to eight lymphocytes per macrophage on the cover slip surface. Macrophages were incubated in vitro 1 h before lymphocyte addition. Cultures were incubated statically at 370C in a 5% C02-95% air atmosphere. Quantitation of the response. At various times (generally 1, 24, and 48 h), phase-contrast, light-microscopic observations of living cultures were carried out. Cells in an area containing approximately 50 macrophages were counted on each of three cover slips, and the number of lymphocytes attached and unattached to macrophages were determined. The attached lymphocytes were subdivided into the percent centrally attached and the percent attached to the peripheral areas of the macrophages. Cells from sensitized mice. Eight- to 10-week-old female Swiss-Webster ROR outbred mice were injected intravenously with 0.1 ml of a suspension, 5 x 107 cells per ml, of living Brucella abortus (strain 19Weybridge) as previously reported (56). It was found that at 21 days postinoculation, these mice exhibited strong delayed-type hypersensitivity to brucella antigens as measured by both the footpad test and migration inhibitory factor (MIF) production in response to B. abortus antigens (56). In addition, these mice have been shown to have an increased ability to destroy faculative intracellular parasites (18). Seven days before collection of peritoneal cells, the mice received a 0.1-ml intraperitoneal injection of a 2,500-,ug/ml suspension of crude sonic extract of phenol-killed B. abortus (strain 2308) prepared as reported previously (21). Lymphocytes and peritoneal macrophages obtained from these animals were processed as before; autologous spleen or thymus cells separated by FicollHypaque were added to antigen-sensitized macro-

255

phage monolayers in both the presence and the absence of 250 jig of B. abortus antigen per ml. In vivo cultures. Washed, sterile, round glass cover slips (12 mm in diameter) were surgically inserted into peritoneal cavities of 8- to 12-week-old female Swiss-Webster ROR mice. Cover slips were removed at various times up to 10 months postimplantation, fixed immediately on removal, and prepared for scanning electron microscopy exactly as for in vitro cultured cells. MIF. MIF was prepared as described previously (58). Briefly, spleen cell suspensions from either Brucella-infected or uninfected female Swiss-Webster ROR mice were incubated in medium NCTC-109 with B. abortus antigen at 37°C in a 5% C02-95% air atmosphere for 24 h. The supernatant fluid was collected, centrifuged to remove cells and cell debris, filtered through a microporous filter (0.45-am approximate pore diameter), and concentrated five times by way of ultrafiltration (Millipore Pellicon filter; nominal molecular weight limit, 1,000). Fractions containing MIF activity were separated via preparative gel electrophoresis (Fractophorator, Buchler Instruments, Inc.), dialyzed against distilled water, and sterilized by filtration. The fractions were then heated at 56°C for 30 min to inactivate complement. Various fractions were assayed for MIF activity, using a capMiary migration assay, and for macrophage spreading activity. A fraction containing MIF as well as macrophage spreading factor (MSF) was obtained (similar fractions from supernatants of spleen cells taken from uninfected mice and cultured with antigen, or cells of infected mice cultured without antigen, showed no MIF or MSF activity). The fraction was then concentrated via evaporation and reconstituted in phosphatebuffered saline to give an optical density of 0.9 at 280 nm. Normal peritoneal macrophages in combination with spleen, thymus, or peritoneal lymphocytes were cultured in dishes containing 1.8 ml of NCTC-109 plus 0.2 ml of phosphate-buffered saline containing MIFMSF. Controls consisted of only NCTC-109 and phosphate-buffered saline. Scanning electron microscopy. Cover slips bearing cells were removed at various times during the course of incubation and placed in a phosphatebuffered 1.5% glutaraldehyde fixative (pH 7.2, 320 total mosmol, including fixative). Fixation was allowed to proceed at 37°C for 48 h with one change of fixative at 24 h. Cells on cover slips were then alcohol dehydrated to 100% ethanol, which was replaced by 100% amyl acetate as the intermediate fluid prior to critical-point dehydration, utilizing CO2 as the transitional fluid. The cover slips with the adhering dehydrated specimens were cemented to metal stubs and then coated with 100% gold to a thickness of 15 to 30 nm in a vacuum evaporator with the aid of a constantly rotating and tilting apparatus (2). Specimens were stored at 0% relative humidity before examination at 10 to 20 kV with a Cambridge scanning electron microscope, Mark II. Transmission electron microscopy. Cells on cover slips were fixed as for scanning electron microscopy, ethanol dehydrated, and embedded in epoxy blocks (Epon), utilizing inverted Beem capsules. After polymerization the blocks were "peeled" from the

256

ALBRECHT ET AL.

slips, and thin sections were poststained in lead citrate and uranyl acetate and examined on a Phillips 300 transmission electron microscope. Immunofluorescence. Goat anti-mouse immunoglobulin antiserum was obtained from Hyland Laboratories (Costa Mesa, Calif.); utilizing indirect immunofluorescence, this antiserum stained 44% of spleen cells and less than 5% of thymocytes obtained from BALB/c mice. Anti-mouse theta (brain-associated) antiserum was prepared in rabbits by the method of Golub (16) and showed, by indirect immunofluorescence, membrane staining with greater than 95% of the thymocytes and 48% of the spleen cells from BALB/c mice. Phagocytosis. A 0.05-ml suspension of 0.81-,imdiameter latex beads (Difco, Detroit, Mich.) was allowed to settle onto cover slips containing 24- or 48-h cultures of macrophages with or without added lymphocytes. Cultures were incubated at 37°C. At various intervals up to 2 h, cells were examined via light microscopy for the presence of phagocytized latex particles.

cover

RESULTS

Lymphocyte-macrophage interaction in vitro. Observations of mouse spleen, thymus, or peritoneal lymphocytes incubated in standing cultures with syngeneic macrophages initially showed a limited degree of lymphocyte-macrophage contact (Fig. la). However, by 24 h numerous lymphocytes could be seen on peripheral areas of nearly all macrophages (Fig. lb). In addition, macrophage processes, often three to four in number and up to 30 ,tm in length, could be seen to extend from the macrophages to the more distant lymphocytes; lymphocytes were often observed to be attached at different points along these macrophage processes. Occasionally, when two or more lymphocytes were in close proximity to a single macrophage process, separate extensions of the same process were seen to contact each lymphocyte (Fig. 2). Initial contact appeared to be made by macrophage microprocesses, as small as 0.25 ,um in diameter, which extended from the cell body or from cell ptocesses.

After initial attachment, the lymphocytes often observed to cluster over the more central areas of the macrophage (Fig. 3a and b). During this period, both smooth and villous peritoneal or spleen lymphocytes could be seen in close physical approximation in these clusters (Fig. 4a and b). Lymphocytes with no villi, a low bumpy surface, or few villi (less than 20) were classed as smooth. In the case of thymic lymphocyte clusters, all attached cells appeared smooth (Fig. 5). The lymphocytes adhered firmly and could not be detached by vigorous agitation of the tissue culture fluid. Uropod rosettes, previously reported in certain guinea pig

were

INFECT. IMMUN.

lymphocyte-macrophage cultures (44), were not seen. In addition, macrophages with lymphocytes clustered on their surfaces retained their phagocytic properties and were observed to actively ingest latex spheres during lymphocyte attachment and cluster formation. Lymphocyte clusters were not regularly observed other than on macrophage surfaces. Transmission electron microscopy after lymphocyte attachment showed some areas of macrophage-lymphocyte membrane contact, with no apparent specialization of the cytoplasm beneath the sites of attachment. In other cases, more extensive areas of contact were observed, with some apparent subsurface modification seen in the macrophage cytoplasm. Kinetic studies. Kinetic studies with syngeneic BALB/c macrophages and lymphocytes showed an initial attachment of 21 ± 2% of the thymocytes to macrophages at 1 h, increasing to 62 ± 6% by 24 h and 88 ± 5% by 48 h. Spleen lymphocytes showed an initial attachment of 33 ± 12%, increasing to 75 ± 5% at 24 h and 87 ± 12% at 48 h (Fig. 6a). This corresponds to an absolute number of zero to two lymphocytes per macrophage, initially, increasing to seven to ten lymphocytes per macrophage by 48 h. Centrally attached thymocytes (percentage of the attached lymphocytes clustered over the central area of the macrophage body) increased over 48 h (17 ± 13% at 1 h, 40 ± 13% at 24 h, 72 ± 6% at 48 h). The percentage of spleen lymphocytes centrally attached was 3 ± 3% at 1 h, increasing to 10 ± 0% at 24 h and 51 ± 6% at 48 h (Fig. 6b). Viability of cells. The percentage of live cells as demonstrated by trypan blue exclusion was greater than 95% for macrophages at 1, 24, and 48 h. Viability was greater than 95% at 1 h, 75 to 80% at 24 h, and 65 to 70% at 48 h for spleen or thymus lymphocytes. Dead lymphocytes were observed on normal unstimulated macrophages and in central clusters after 24 to 72 h of culture; however, their proportion was equal to or less than the percentage of dead cells when lymphocytes were cultured in the absence

of macrophages. Many lymphocyte clusters on macrophages had no dead lymphocytes even after 96 h of culture. Attachment of fibroblasts. Thymocytes present in cultures of thymic fibroblasts or added after establishment of a thymic fibroblast monolayer failed to attach to or cluster on the fibroblasts to any significant extent. Clusters of lymphocytes were, however, attached to macrophages which had migrated from the original thymic piece and could be found at the periphery of the fibroblast monolayer.

.t

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

4~

~

K

~

o

t-c

.s..

.0--

~

n

-

257

_-0

v . -.ot ~' v

cS i,~ ?£$ ;0 OC_ -,. . # 'l;o n

-

,

M

-

ft- .

=,: s-

f-

41

-

F

iO

>

~O.f 0 O

,

d F_

_

>_;X

g

w

_

-.,;i ~^- !

--r.

FIG. 1. Light microscopy of normal peritoneal macrophages and syngeneic thymocytes after 10 min (a) and 24 h (b) of incubation. Small round cells are lymphocytes; larger rounded cells are unspread macrophages; very small irregular round cells are platelets which can be seen free, attached to macrophages and occasionally to lymphocytes. x200.

258

ALBRECHT ET AL.

INFECT. IMMUN.

FIG. 2. Peritoneal macrophage with attached autologous smooth (S) and villous (V) lymphocytes after 24 h of culture. Attachment is to macrophage peripheral areas and cell processes. x3,050.

Fetal calf serum. The addition of fetal calf serum to a final concentration of 1 or 5% in the cultures did not inhibit the initial 1-h attachment but precluded any significant attachment and clustering during the ensuing 48-h incubation period. The percentage of splenic or thymic lymphocytes attached to macrophages was 30 to 40% at 1 h and remained at that level or had decreased by 48 h. The number of centrally attached lymphocytes remained less than 10%, except for the thymus cells at 48 h, which showed a slight increase (Table 1). No significant difference in cell viability, as assessed by trypan blue, was noted in cultures with or without fetal calf serum. Effect of MIF. Peritoneal macrophages cultured with peritoneal, spleen, or thymus lymphocytes in the presence of the MIF-MSF-containing fraction spread more readily than normal controls. Sixty percent of all macrophages grown with the MIF-MSF fraction were well spread by 6 h as compared to 10% of control cells spread by this time. In addition, many of the spread macrophages had lymphocytes attached at their periphery, whereas control macrophages had

few attached lymphocytes. Subsequent lymphocyte clustering appeared to follow normal parameters as described earlier, and no effect other than the acceleration of macrophage spreading and concomitant lymphocyte attachment was observed. Attachment to antigen-activated macrophages. Peritoneal macrophages from Brucella-infected mice cultured in vitro with thymocytes from the same animal showed large numbers of flattened, extensively spread macrophages with numerous peripheral processes. These macrophages lacked extensive ruffled membranes and had primarily thymocytes on their surfaces; however, other round cells, possibly macrophages or neutrophils, were also present. By 8 h, greater than 99% of all lymphocytes were attached to macrophage surfaces. Some lymphocytes appeared to be clustered, but in most instances the lymphocytes were scattered randomly over the macrophage surface, showing little or no lymphocyte-to-lymphocyte contact (Fig. 7a). Similar cells and cell associations were seen in both the presence and absence of 250 [Lg of B. abortus antigen per ml in the

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

259

I ...,, ~ = ME M &16 FIG. 3. (a) Light microscopy of lymphocyte-macrophage clusters after 48 h of culture. (b) Peritoneal macrophages with attached autologous lymphocytes after 48 h of culture. Lymphocytes tend to cluster over the central nuclear area of the macrophage. X1,0O. *

260

ALBRECHT ET AL.

INFECT. IMMUN.

FIG. 4. (a) Villous-smooth and smooth-smooth lymphocyte contact in clustered syngeneic splenic lymphocytes. Macrophage surface is in background. x12,200. (b) Villous-villous lymphocyte contact in clustered syngeneic splenic lymphocytes. X12,500.

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

261

FIG. 6. Syngeneic thymnocytes on macrophage surface. xlO,100.

culture medium. At 24 h, lymphocytes which remained attached to macrophage surfaces developed a spongy outer surface indicative of dead or dying cells. Rounded macrophages on the surface of the spread macrophages also appeared to be dead or dying by 24 h of incubation (Fig. 7b). Macrophages from normal uninfected mice with and without antigen in the media appeared normal, and added thymocytes attached and clustered as described previously. Allogeneic and xenogeneic studies. The addition of allogeneic Swiss-Webster ROR thymocytes or xenogeneic human thymocytes to BALB/c macrophages resulted in an initial attachment of thymocytes to peripheral areas of the macrophage cell body; however, subsequent clustering failed to occur even by 72 h (Fig. 8). By 48 h, 80% of allogeneic thymocytes were adherent to macrophages, although only 14% appeared centrally attached; 51% of xenogeneic lymphocytes were attached, with 36% of those attached centrally located. Controls utilizing syngeneic thymocytes showed 88% of the lymphocytes attached to macrophages, with 72% of these being centrally attached by 48 hours. No phagocytosis of allogeneic or xenogeneic lymphocytes was observed even after 72 h of incubation with macrophages (Table 2).

In vivo lymphocyte-macrophage interactions. In vivo macrophage-lymphocyte combinations were also observed. Cover slips removed up to 10 months postimplantation were found to be covered by adherent macrophages exhibiting varying degrees of spreading, as well as pseudopodia, membrane ridges, ruffles, and villi. Lymphocytes (both villous and smooth) were seen singly and in clusters over central areas of macrophage cell bodies, as well as attached to cell margins or processes (Fig. 9). Lymphocytes not attached to adherent cells were rarely seen. DISCUSSION Our findings suggest that the lymphocytemacrophage interaction involves initial contact of lymphocytes with macrophage cell bodies or processes followed by events leading to lymphocyte clustering on macrophage surfaces. Nearly all adherent macrophages from unstimulated peritoneal cavities can serve as sites for binding and clustering of syngeneic lymphocytes in vitro. In vitro, nearly all lymphocytes are able to bind to macrophages in the absence of sera, irritants, or antigens to which the lymphocytes or macrophages have been sensitized. Previous studies, also in an antigen-independ-

262

ALBRECHT ET AL.

INFECT. IMMUN. ent system, with syngeneic guinea pig peritoneal

exudate macrophages and lymph node or thymic lymphocytes showed less binding. If lymph node lymphocytes were added to ratios of 50:1 lymphocytes/macrophage, 1.4% of all lymphocytes were bound in rocking culture at 1 h (32). Thymic lymphocytes also showed similar low levels of binding, in static culture or when peri-

a

-p@, ......

4

toneal exudate macrophages were cultured in vitro only 1 h before lymphocyte additions. Higher rates of binding could be obtained with decreased ratios of thymocytes to macrophages or when macrophages were cultured 24 h in vitro

MMM8M"

before the addition of

Ib I g.

-,^,,* at

8

16

24

lymphocytes (31, 32).

In

the present study, approximately 20% of the spleen or thymic lymphocytes were bound in 1 h to normal (as opposed to exudate) mouse peritoneal macrophages cultured in vitro only 1 h before lymphocyte addition. Binding increased to nearly 90% after 48 h of incubation. Differences in animal species, lymphocyte source, or technique may be causes for different observations; the results may also reflect functional differences between normal peritoneal as opposed to exudate macrophages. Evidence suggests the peritoneal macrophage may be a selfperpetuating subpopulation of cells, whereas peritoneal exudate macrophages are derived primarily from migration and maturation of blood monocytes (61). The mechanisms of lymphocyte movement

the macrophage surface which lead to cenare unexplained. Both lymphocyte motility (35, 36) and propulsive waves of the macrophage plasma membrane which move centrally from peripheral areas may be involved over

HOM

FIG. 6. (a) Percentage of BALB/c thynnocyte and spleen lymphocytes attached to BALB,'/c macrophages after 1, 24, and 48 h of culture. Mrean of five separate experiments ± 2 standard devilnations. (b) Percentage of BALB/c thymocytes and spleen lymphocytes clustered over the central nuclear area of BALBIc macrophages in contrast to thos-e attached to peripheral areas or cell processes after 1, 24, and 48 h of culture. Mean of five separate expieriments ± 2 standard deviations.

tral clustering

(9).

Splenic and thymic lymphocytes seem to bind equally well to macrophages. Since nearly 90% of splenic lymphocytes (approximately half of which are T cells and half E cells) adhere to

TABLE 1. Percentage of lymphocytes attached to and clustered over the central area of macrophages in the presence and absence of fetal calf serum (FCS) Parameter

Attached to macrophages Clustered over central area of macrophages

Time after addition of lymphocytes

Lymphocytes (%)

Spleen

Thymus

(h)

0% FCSa

1% FCSb

5% FCSb

0% FCSa

1% FCSb

5% FCSb

1

33 75 87

39

35 19 19

21 62 88

24 45 28

31 24 17

17 40 72

0 7 10

3 0 17

24 48

41

24

1 3 9 7 24 10 0 0 48 51 6 0 a Mean of five experiments, three separate determinations per experiment. b Mean of two experiments, three separate determinations per experiment.

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

263

FIG. 7. (a) Swiss- Webster ROR thymocytes and autologous peritoneal macrophages from B. abortusinfected animals after 8 h in culture with 250 pg of B. abortus antigen per ml. xl, 100. (b) Swiss- Webster ROR thymocytes and autologous macrophages from B. abortus-infected mice after 24 h in culture with 250 pg of B. abortus antigen per ml. x6,600.

264

INFECT. IMMUN.

ALBRECHT ET AL.

FIG. 8. Allogeneic ROR thymocytes in culture with BALBIc macrophages for 72 h. Note the absence of cells over the central nuclear area. x3,200. TABLE 2. Lymphocyte attachment in syngeneic, allogeneic, and xenogeneic cell combinations" Lymphocytes (%)

Cell combination

PCM, + TM,

PCM, + TM2 PCM, + TH

Attached

Centrally attached

24 h

48 h

24 h

48 h

62 76

88 80 51

34 18 17

72 14 36

73

a Average of two experiments, each consisting of triplicate determinations.

POM,,

b Normal peritoneal macrophages, BALB/c mouse; TM,, thymocytes, BALB/c mouse; TM2, thymocytes, ROR mouse; TH, human thymocytes.

macrophages (48), it seems likely that both B and T lymphocytes are associated with macrophages, as has been reported by Lipsky and Rosenthal (32). Preliminary immunofluorescent studies show both immunoglobulin-positive and theta-positive cells binding to macrophages in central clusters (Albrecht et al., Program Abstr. 9th Leukocyte Culture Conf., 1974. Abstr. no. 6). A greater proportion of thymic lymphocytes appear able to cluster centrally. The functional significance, if any, of the smooth as opposed to

villous surface morphology of lymphocytes clustered on macrophage surfaces has yet to be determined. Previous scanning and transmission electron microscopic studies have suggested that villous and smooth cells (in mice and humans) are the equivalents of B and T lymphocytes, respectively (46, 48, 50). However, care must be exercised in the interpretations of these results, since other reports show smooth and villous cells not to be associated with any particular lymphocyte subpopulations (3, 22, 30, 39). The reasons for these divergent observations are not clear, although the mode of cell collection and preparation before scanning electron microscopy has been shown to influence the ratio of smooth to villous cells (3, 6, 19, 28, 29, 43). The degree of maturation of cells, the presence of certain lymphocyte-erythrocyte rosettes, and the species of animals used may also influence the lymphocyte surface structure (3, 10, 22, 23, 27, 29, 47, 67). Hence, lymphocyte surface morphology may relate to the degree of maturation and/or reflect functional characteristics. Ultimately this may be of greater significance than whether such cells correlate with what is presently defined as a "B" or "T" lymphocyte. Addition of 1 or 5% fetal calf serum partially

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

265

FIG. 9. Intraperitoneally implanted cover slips removed after 7 days in vivo. Macrophages with attached lymphocytes have spread over the cover slip surface. x1,500.

inhibits the response seen in serum-free unstimulated syngeneic systems. Similar initial binding rates are present; however, additional binding and clustering appear to be inhibited, and some bound lymphocytes appear to be released. Similar findings have been described by Lipsky and Rosenthal for the binding of guinea pig thymocytes to syngeneic peritoneal exudate macrophages in the presence of 10% fetal calf serum (31, 33). The basis for this effect is not clear and may be a nonspecific serum effect on macrophage membranes, although activation of some macrophage receptor sites, e.g., guinea pig macrophage Fc receptor, has been shown to be inhibited by both normal and heat-inactivated guinea pig sera (51). The increase in macrophage spreading due to the MIF-containing fractions is similar to previously reported results (42, 57, 58). In addition, it appears as though this more rapid spreading occurs in the presence of spleen, thymic, or peritoneal-cavity lymphocytes and leads to an earlier attachment of the lymphocytes to the macrophages. Thus, MIF may not only result in macrophages remaining in an area of inflammation but may also serve directly or indirectly to accelerate the interaction of lymphocytes with macrophages and hence subsequent lympho-

cyte-to-lymphocyte interactions on macrophage surfaces. The use of antigen-sensitized peritoneal macrophages from infected syngeneic mice in our serum-free system resulted in a more rapid binding of lymphocytes to macrophages whether or not antigen was present during incubation. Increased macrophage spreading as has been reported for antigen-activated macrophage systems was observed (12); however, in many instances lymphocytes attached to macrophages appeared to be dying by 24 h, perhaps due to the release of a cytotoxic factor(s) by the highly activated macrophages (11, 53). The apparent death of these attached lymphocytes and the absence of central lymphocyte clusters on macrophages seen in this antigenactivated system could be the basis for the depression of splenic lymphocyte responsiveness to phytohemagglutinin, concanavalin A, and bacterial lipopolysaccharide observed in Corynebacterium parvum-activated systems. Both the inhibition of tumor cell growth and the decreased spleen lymphocyte mitogen responsiveness seen with C. parvum activation have been reported to be due to the presence of the highly activated macrophages (24). Binding of thymocytes to normal nonacti-

266

ALBRECHT ET AL.

vated macrophages occurs with syngeneic as well as allogeneic and xenogeneic cells. A reduction in the efficiency of binding for allogeneic and xenogeneic thymocytes has been reported by others (34); however, in this study and in the previously described system reported by Lipsky and Rosenthal (33), no difference was noted in the initial binding efficiency of syngeneic versus allogeneic or xenogeneic lymphocytes. None of the adhering syngeneic, allogeneic, or xenogeneic lymphocytes are phagocytosed, although the macrophages with associated lymphocytes actively phagocytose latex particles; similar "segmental" responses of macrophage membranes have been reported previously (17). Allogeneic and xenogeneic lymphocytes remain peripheral or paranuclear, whereas syngeneic cells tend to cluster over the nuclear area. The possibility of shearing forces during preparation procedures has been suggested as a reason for the absence of binding of cells over the central raised nuclear area (45, 65). However, in the present study, light-microscopic observations during culture, before any washing, show no centrally attached allogeneic or xenogeneic cells; also, the presence of centrally located cell clusters in the syngeneic systems tends to rule out shear forces. The apparent restriction of xenogeneic and allogeneic thymocytes to peripheral and perinuclear areas may be of important functional significance, since previous studies have shown the need for histocompatible (specifically, Ia-region identity) thymocytes and macrophages in the in vitro generation of T helper cells to nonparticulate antigen in mice (11) and in antigen-induced, T-lymphocyte activation in guinea pigs (53, 54). ACKNOWLEDGMENTS We thank Richard Hong for his helpful advice and review of this manuscript. We also thank Thomas Barber for his assistance in preparation and observation of transmission electron microscopic samples and S. Keller, G. Albrecht,,and J. Albrecht for their technical assistance. LITERATURE CITED 1. Albrecht, R., R. Hinsdill, P. Sandok, A. MacKenzie, and I. Sachs. 1972. A comparative study of the surface morphology of stimulated macrophages prepared without chemical fixation for scanning EM. Exp. Cell. Res. 70:230-232. 2. Albrecht, R. M., and A. P. MacKenzie. 1975. Cultured and free living cells, p. 109-153. In M. A. Hayat (ed.), Principles and techniques of scanning electron microscopy: biological applications, vol. 3. Van Nostrand Reinhold Co., New York. 3. Alexander, E. L., and B. Wetzel. 1975. Human lymphocytes: similarity of B and T cell surface morphology. Science 188:732-734. 4. Askonas, B., and G. Roelants. 1974. Macrophages bearing hapten-carrier molecules as foci inducers for T and B lymphocyte interaction. Eur. J. Immunol. 4:1-4.

INFECT. IMMUN. 5. Auerbach, R. 1971. Towards a developmental theory of immunity: cell interactions, p. 393-398. In 0. Makela, A. Cross, and T. U. Kosunen (ed.), Cell interactions and receptor antibodies in immune responses. Academic Press Inc., New York. 6. Barber, T. A., and P. M. Burkholder. 1975. Relation of surface and internal ultrastructure of thymus and bone marrow derived lymphocytes to specimen preparatory technique, p. 369-378. In 0. Jahari and I. Corvin (ed.), Scanning electron microscopy. IITRI, Chicago. 7. Biniaminov, M., B. Ramot, E. Rosenthal, and A. Novogrodsky. 1975. Galactose oxidase-induced blastogenesis of human lymphocytes and the effect of macrophages on the reaction. Clin. Exp. Immunol. 19:93-98. 8. Chused, Y., and R. Andersen. 1974. Studies on the long term growth of macrophages obtained from various sources, p. 5-14. In W. Wagner, H. Hahn, and R. Evans (ed.), Activation of macrophages. American Elsevier Publishing Co., Inc., New York. 9. Cohn, Z. 1975. Macrophage physiology. Fed. Proc. 34:1725-1729. 10. Criswell, B. S., R. R. Rich, J. Dardano, and S. L. Kimzey. 1975. Scanning electron microscopy of normal and mitogen-stimulated mouse lymphoid cells. Cell. Immunol. 19:336-348. 11. Erb, P., and M. Feldman. 1975. The role of macrophages in the generation of T-helper cells. II. The genetic control of the macrophage-T-cell interaction for helper cell induction with soluble antigens. J. Exp. Med. 142:460-472. 12. Evans, R. 1974. Specific and non-specific activation of macrophages, p. 305-317. In W. Wagner, H. Hahn, and R. Evans (ed.), Activation of macrophages. American Elsevier Publishing Co., Inc., New York. 13. Evans, R., and P. Alexander. 1972. Role of macrophages in tumor immunity. I. Cooperation between macrophages and lymphoid cells in syngeneic tumor immunity. Immunology 23:615-626. 14. Gallily, R., and Z. Ben-Ishay. 1974. Interaction between normal or irradiated macrophages and lymphocytes in mice. Cell. Immunol. 11:314-324. 15. Gisler, R., and P. Dukor. 1972. A three-cell mosaic culture: in vitro immune response by a combination of pure B- and T-cells with peritoneal macrophages. Cell. Immunol. 4:341-350. 16. Golub, E. S. 1971. Brain-associated antigen reactivity of rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2:353-361. 17. Griffin, F., and S. Silverstein. 1974. Segmental response of the macrophage plasma membrane to a phagocytic stimulus. J. Exp. Med. 139:323-336. 18. Halliburton, B. L., and R. D. Hinsdill. 1972. Recall of acquired cellular resistance in mice by antigens from killed Brucella. Infect. Immun. 6:42-47. 19. Hammerling, V., A. Polliack, N. Lampen, M. Sabety, and E. de Harvin. 1975. Scanning electron microscopy of tobacco mosaic virus-labelled lymphocyte surface antigens. J. Exp. Med. 141:518-523. 20. Hanifin, J. M., and M. J. Cline. 1970. Human monocytes and macrophages (interaction with antigen and lymphocytes). J. Cell. Biol. 46:97-105. 21. Hinsdill, R. D., and D. T. Berman. 1967. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic characterization. J. Bacteriol. 93:544-549. 22. Hirsch, M., B. Zisman, and A. Allison. 1970. Macrophages and age-dependent resistance to herpes simplex virus in mice. J. Immunol. 104:1160-1165. 23. Kay, M. M., B. Belohradsky, K. Yee, J. Vogel, D. Butcher, J. Wydran, and H. H. Fudenberg. 1974. Cellular interactions: scanning electron microscopy of human thymus-derived rosette-forming lymphocytes. Clin. Immunol. Immunopathol. 2:301-309.

VOL. 21, 1978

MURINE MACROPHAGE-LYMPHOCYTE INTERACTIONS

24. Kirchner, H., H. T. Holden, and R. B. Heberman. 1975. Splenic supressor macrophages induced in mice by injection of Corynebacterium parvum. J. Immunol. 115:1212-1216. 25. Klaus, G. 1974. Generation of thymus-derived helper cells by macrophage-associated antigen. Cell. Immunol. 10:483-488. 26. Lilien, J. E. 1969. Toward a molecular explanation for specific cell adhesion, p. 169-195. In A. A. Moscona and A. Monroy (ed.), Current topics in developmental biology. Academic Press Inc., New York. 27. Lin, P. S., A. G. Cooper, and H. H. Wortis. 1973. Scanning electron microscopy of human T-cell and Bcell rosettes. N. Engl. J. Med. 289:548-551. 28. ]Un, P. S., D. F. H. Wallach, and S. Tsai. 1973. Temperature-induced variations in the surface topology of cultured lymphocytes are revealed by scanning electron microscopy. Proc. Natl. Acad. Sci. U.S.A. 70:2492-2496. 29. Linthicum, D. S., and S. Sell. 1974. A rabbit blood lymphocyte is both smooth and hairy. N. Engl. J. Med. 290:1327-1328. 30. Linthicum, D. S., S. Sell, R. M. Wagner, and P. Trefts. 1974. Scanning immunoelectron microscopy of mouse B and T lymphocytes. Nature (London) 252:173-175. 31. Lipsky, P., and A. Rosenthal. 1973. Macrophage-lymphocyte interaction. I. Characteristics of the antigenindependent-binding of guinea pig thymocytes and lymphocytes to syngeneic macrophages. J. Exp. Med. 138:900-924. 32. Lipsky, P., and A. Rosenthal. 1975. Macrophage-lymphocyte interaction: antigen-independent binding of guinea pig lymph node lymphocytes by macrophages. J. Immunol. 115:440-445. 33. Lipsky, P., and A. Rosenthal. 1975. Macrophage-lymphocyte interaction. II. Antigen-mediated physical interactions between immune guinea pig lymph node lymphocytes and syngeneic macrophages. J. Exp. Med. 141:138-154. 34. Lopez, L. R., K. S. Johansen, J. Radovich, and D. W. Talmage. 1974. Thymocytes, macrophages and erythrocytes. J. Allergy Clin. Immunol. 53:336-344. 35. Matthes, M., W. Ax, and H. Fischer. 1971. Microcinematographic analyses of the cooperation between lymphocytes and dendritic macrophages following primary and secondary stimulation with soluble antigen, p. 15-23. In 0. Makela, A. Cross, and T. U. Kosunen (ed.), Cell interactions and receptor antibodies in immune responses. Academic Press Inc., New York. 36. Matthes, M., W. Ax, and H. Fischer. 1971. Mikrokinematographische Studien uber die Zellcooperation von Lymphocytes und Makrophagen nach primarem und sekundarem Stimulus mit loslichem Antigen. Z. Gesamte Exp. Med. 154:253-264. 37. Melsom, H., and R. Seljelid. 1973. The cytotoxic effect of mouse macrophages on syngeneic and allogeneic erythrocytes. J. Exp. Med. 137:807-820. 38. Miller, H. R. P., and S. Avrameas. 1971. Association between macrophages and specific antibody producing cells. Nature (London) New Biol. 229:184-185. 39. Molday, R. S., W. J. Dreyer, A. Rembaum, and S. P. S. Yen. 1975. New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy. J. Cell. Biol. 64:75-88. 40. Moscona, A. A. 1960. Recombination of dissociated cells and the development of cell aggregates in cells and tissues in culture, p. 489-529. In E. N. Wilmer (ed.), Cells and tissues in culture methods, biology, and physiology, vol. 1. Academic Press Inc., New York. 41. Mosier, D. 1969. Cell interactions in the primary immune response in vitro. A requirement for specific cell clusters. J. Exp. Med. 129:351-362. 42. Nathan, C. F., H. G. Remold, and J. G. David. 1973. Characterization of a lymphocyte factor which alters

267

macrophage functions. J. Exp. Med. 137:275-290. 43. Nemanic, M. K., D. P. Carter, D. R. Pitelka, and L. Wofsy. 1975. Hapten-sandwich labelling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy. J. Cell. Biol. 64:311-321. 44. Nielsen, M., H. Jensen, 0. Braendstrug, and 0. Werdelin. 1974. Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. II. Ultrastructure of clusters formed during the early response. J. Exp. Med. 140:1260-1272. 45. Papadimitriou, J. M. 1973. Detection of macrophage receptors for heterologous IgG by scanning and transmission electron microscopy. J. Pathol. 110:213-220. 46. Polliack, A., and E. de Harvin. 1975. An interpretative review surface features of normal and leukemic lymphocytes as seen by scanning electron microscopy. Clin. Immunol. Immunopathol. 3:412-430. 47. Polliack, A., S.-M. Fu, S. D. Douglas, Z. Bentwich, N. Lampin, and E. de Harvin. 1974. Scanning electron microscopy of human lymphocyte rosettes. J. Exp. Med. 140:146-158. 48. Polliack, A., V. Hammerling, N. Lampin, and E. de Harvin. 1975. Surface morphology of murine B and T lymphocytes: a comparative study by scanning electron microscopy. Eur. J. Immunol. 5:32-39. 49. Remington, J., J. Krahenbuhl, and J. Hibbs. 1975. A role for the macrophage in resistance to tumor development and tumor destruction, p. 869-893. In E. van Furth (ed.), Mononuclear phagocytes in immunity, infection and pathology. Blackwell Scientific Publications Ltd., Oxford. 50. Reyes, F., J. L. Lejonc, M. F. Gourdin, P. Munnoni, and B. Dreyfus. 1975. The surface morphology of human B lymphocytes as revealed by immunoelectron microscopy. J. Exp. Med. 141:392-410. 51. Rhodes, J. 1975. Macrophage heterogeneity in receptor activity: the activation of macrophage Fc receptor function in vivo and in vitro. J. Immunol. 114:976-981. 52. Rode, H., and J. Gordon. 1974. Macrophages in the mixed leukocyte culture reaction (MLC). Cell. Immunol. 13:87-94. 53. Rosenthal, A., P. Lipsky, and E. Shevach. 1975. Macrophage-lymphocyte interaction and antigen recognition. Fed. Proc. 34:1743-1748. 54. Rosenthal, A., and E. Shevach. 1973. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J. Exp. Med. 138:1194-1212. 55. Salvin, S., S. Sell, and J. Nishio. 1971. Activity in vitro of lymphocytes and macrophages in delayed hypersensitivity. J. Immunol. 107:655-662. 56. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht. 1971. Migration inhibition of mouse macrophages by BruceUa antigens. Infect. Immun. 4:516-518. 57. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht. 1974. Isolation of substances responsible for lymphokine activity from sensitized spleen cells. Infect. Immun. 9:1045-1050. 58. Sandok, P. L., R. D. Hinsdill, and R. M. Albrecht. 1975. Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation, and fractionation. Infect. Immun. 11:1100-1109. 59. Schoenberg, M. D., V. R. Mumaw, R. D. Moore, and A. S. Weisberger. 1964. Cytoplasmic interaction between macrophages and lymphocytic cells in antibody synthesis. Science 143:964-965. 60. Seeger, R., and J. Oppenheim. 1970. Synergistic interaction of macrophages and lymphocytes in antigen-induced transformation of lymphocytes. J. Exp. Med. 132:44-65. 61. Shands, J. W., and B. J. Axelrod. 1977. Mouse peritoneal macrophages: tritiated thymidine labelling and cell

268

ALBRECHT ET AL.

kinetics. RES J. Reticuloendothel. Soc. 21:69-76. 62. Sharp, J. A., and R. G. Burwell. 1960. Interaction ('peripolesis') of macrophages and lymphocytes after skin homografting or challenge with soluble antigens. Nature (London) 188:474-475. 63. Siegel, I. 1970. Autologous macrophage-thymocyte interactions. J. Allergy 46:190-194. 64. Sulitzeanu, D. R., R. Kleinman, D. Benezra, and I. Gery. 1971. Cellular interactions and the secondary response in vitro. Nature (London) New Biol. 229:254-255. 65. Tizard, I. R., W. L. Holmes, and N. P. Parappally. 1974. Phagocytes of sheep erythrocytes by macrophages: a study of the attachment phase by scanning electron microscopy. RES J. Reticuloendothel. Soc. 15:225-231.

INFECT. IMMUN. 66. Unanue, E., and D. Katz. 1973. Immunogenicity of macrophage-bound antigens: the requirement for hapten and carrier determinants to be on the same molecule for T and B lymphocyte collaboration. Eur. J. Immunol. 3:559-563. 67. van Ewijk, W., N. H. C. Brons, and J. Rozing. 1975. Scanning electron microscopy of homing and recirculating lymphocyte populations. Cell. Immunol. 19:245-261. 68. Yoshinaga, M., S. Nakamura, and H. Hayashi. 1975. Interaction between lymphocytes and inflammatory exudate cells. I. Enhancement of thymocyte response to PHA by product(s) of polymorphonuclear leukocytes and macrophages. J. Immunol. 115:533-538. 69. Zembala, M., W. Ptak, and M. Hancyakowska. 1973. Macrophage and lymphocyte cooperation in target cell destruction in vitro. Clin. Exp. Immunol. 15:461-465.