Clin. exp. Immunol. (1979) 35, 258-268.

Human monocyte-lymphocyte interaction and its enhancement by levamisole I. W. KAZURA, W. NEGENDANK, D. GUERRY & A. D. SCHREIBER Hematology-Oncology Sectiont, University ofPennsylvania School oJ'Medicine, Philadelphia, Pennsylvania, USA (Received 9 May 1978)

SUMMARY

We investigated the role of monocyte-lymphocyte interaction in the transformation of human peripheral blood lymphocytes by the mitogen concanavalin A (Con A). Human monocytes were separated from lymphocytes and were transiently exposed to Con A. The Con A-pretreated monocytes were able to subsequently bind autologus lymphocytes by a process that was selective for T cells. This interaction required the initial presence of Con A at the monocyte surface, and became independent of surface bound ligand after 72 hr. Levamisole, an agent thought to facilitate the participation of monocytes in the cellular immune response, enhanced the binding of lymphocytes to monocytes at low concentration of Con A (5-10 Pg/ml). Levamisole did not lead to mitogen independent lymphocyte binding. The association between lymphocytes and Con Apretreated monocytes resulted in the mitogenic transformation of lymphocytes in the absence of soluble Con A in the medium. These results suggest that, in addition to any possible soluble mediators, direct lymphocyte-monocyte contact is required for optimal mitogenic transformation. This T-cell-monocyte interaction over time becomes independent of cell-surface mitogen. The ability of levamisole to enhance this interaction may explain levamisole's capacity to stimulate lymphocyte proliferation. INTRODUCTI ON There is increasing evidence that the monocyte-macrophage is necessary for optimal antigen- and mitogen -induced lymphocyte transformation (Cline & Swett, 1968; Hedfors, Holm & Petterson, 1975; Lohrmann, Novikovs & Graw, 1974; Seeger & Oppenheim, 1970) and that physical interaction between these two cell types occurs during the process of lymphocyte activation (Hannifin & Cline, 1970; Lipscomb, BenSasson & Uhr, 1977). In the guinea-pig, peritoneal macrophages bind T lymphocytes in the absence of antigen or mitogen (Lipsky & Rosenthal, 1973); however, pretreatment of macrophages with priming antigen (Lipsky & Rosenthal, 1975) is necessary to effect lymphocyte transformation. In the present study, we investigated the role of monocyte-lymphocyte interaction in the transformation of human peripheral blood lymphocytes. We observed that human peripheral blood monocytes, unlike guinea-pig macrophages, do not bind syngeneic or allogeneic lymphocytes. When monocytes are briefly exposed to the mitogenic lectin concanavalin A (Con A), however, T-lymphocyte binding occurs and the bound lymphocytes become transformed. Levamisole, a synthetic anti-helminthic agent which is reported to stimulate cell-mediated immunity in man (Pike & Snyderman, 1976; Schreiber & Parsons, 1977; Symoens & Rosenthal, 1977) and increase the lymphocyte proliferative response (Lichtenfeld et al., 1976; Sampson & Lui, 1976), enhances the lymphocyte-monocyte interaction. Correspondence Dr A. D. Schreiber, M.D., Hematology-Oncology Section, Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pa. 19104, USA. 0099-9104/79/0020-0258 $02-00 C 1979 Blackwell Scientific Publications

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MATERIALS AND METHODS Subjects. Blood anticoagulated with heparin (10 units/ml) was obtained by venipuncture from healthy donors between the ages of 20-35 years. Individuals with symptoms of a viral illness within the preceeding 2 weeks were excluded. In each experiment both lymphocytes and monocytes were obtained from the same donor, unless otherwise stated. Monocyte preparation. Monocyte monolayers were prepared by a modification of previously described methods (Schreiber et al., 1975). An equal volume of heparinized blood and 0-15 M NaCl was applied to a Hypaque-Ficoll density gradient and the mononuclear cell suspension was harvested and diluted with HBSS (Hanks's balanced salt solution) (Grand Island Biological, Grand Island, New York) to 2 x 106 or 4x 106 cells/ml. Latex particles, 0-81 pm in diameter (Dow Chemical Co., Indianapolis, Indiana), were added at a final concentration of approximately 107/ml. One ml aliquots of this suspension were placed on 35 mm plastic tissue culture dishes (Falcon Plastics, Oxnard, California) which had been pre-incubated with 1-0 ml of HBSS containing 12.5% autologous serum heat-inactivated at 560C for 30 min (Koller et al., 1973). The cells settled for 1-5 hr at room temperature and those which were non-adherent were removed with five washes of HBSS. Monolayers obtained by this method had > 98% latex-containing mononuclear cells ( > 3 latex particles/cell) which morphologically resembled monocytes when stained with Wright's-Giemsa and examined by light microscopy. Monocyte monolayers were then cultured for 1 hr in 2 ml of F-10 Ham's medium (Microbiological Association, Bethesda, Maryland) containing 100 units of penicillin and 100 jug streptomycin/ml with 20% autologous serum, washed and exposed to Con A for 1 hr (see below). In an attempt to simulate peritoneal macrophages utilized in the guinea-pig model for macrophage-lymphocyte interaction (Lipsky & Rosenthal, 1973), some monocyte monolayers were incubated in culture medium for 42 hr before the addition of Con A. These monocytes morphologically resembled macrophages with a high cytoplasmic: nuclear ratio, 'ruffled' membranes (Bennett & Cohn 1966) and large size, as compared to monocytes cultured for 1 hr. Lymphocyte preparation. A two-step differential adherence technique was utilized to obtain a population of lymphocytes which was not adherent to glass. Mononuclear cell suspensions were obtained by Hypaque-Ficoll density gradient centrifugation and 5 x 106 cells were layered on 35 mm tissue culture plates in 1 ml HBSS for 45 min at room temperature. Non-adherent cells were removed by gentle rinsing with 2 ml of HBSS, sedimented at 250 g, resuspended to 5x 106 cells/ml HBSS and 1 ml layered on tissue culture plates which had been exposed for 10 min to 1 ml of HBSS with 12.5% heat-inactivated autologous serum. After 45 min at room temperature, non-adherent cells were again removed with 2 ml of HBSS, sedimented at 250 g and resuspended to 5 x 106 lymphocytes per ml in VBS (isotonic veronal buffered saline, pH 7*4, containing 0-1% gelatin, 0-00015 M CaC12, and 0-0005 M MgCl2). The yield of mononuclear cells after these two adherence steps was 30%. No further glass adherent cells were detectable, less than 2% of the mononuclear cells were able to phagocytose latex particles, and the cells had the morphological appearance of lymphocytes by light microscopy. This lymphocyte-rich population contained 55% T lymphocytes by the E-rosette method (Jondal, Holm & Wigzell, 1972); 20-25% of the cells rosetted with mouse EAC3 (Mendes et al., 1972). This population of monocyte-depleted lymphocytes exhibited a markedly depressed proliferative response to Con A compared to unfractionated mononuclear cells and to lymphocytes to which monocytes had been added back (Table 1). Exposure of monocytes to Con A. Con A was obtained from Sigma Co. (St. Louis, Missouri) or Miles-Yeda (Elkhart, Indiana). Similar results were obtained with both preparations. One ml of Con A in phosphate-buffered saline pH 7-2 and 1 ml of F-10 Ham's medium containing 1% gelatin were added to washed monocyte monolayers for 1 hr at 37°C in a humid TABLE 1. Proliferative response of mononuclear cell populations

Cell population

Con A (pug/ml) 0 5 10 20

Monocyte-depleted lymphocytes*

Lymphocytes +monocytest

Unfractionated Mononuclear cells

164+20 1476+ 696 2855+ 1239 3429+ 249

141+60 5060+ 1178 7366+ 1432 8474+ 251

175+30 8633+ 1383 15710+ 2274 18093+ 35

*10' lymphocytes were incubated with Con A for 96 hr in 1 ml of RPMI/20% autologous serum and pulsed with 3H-TdR (05 puci) for the last 4 hr of culture. Results are expressed in cpm in trichloroacetic acid precipitable material. The mean+ s.e.m. of triplicate experiments is shown. t105 lymphocytes reconstituted with 104 autologous monocytes, obtained by removal of glass adherent monocytes with 12 mm lidocaine (17). Monocytes alone did not exhibit a proliferative response. G

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atmosphere of 5% CO2 in air. Con A that was not associated with cells was removed by vigorous washing with HBSS, and 1 ml of F-10 Ham's containing 1% gelatin was added. Polymorphonuclear leucocyte suspensions were also obtained from autologous blood (Boyum, 1968) and monolayers were prepared with 2 x 106 leucocytes and pulsed with 10 or 20 ug/ml of Con A. Monocyte-lymphocyte association. One ml of 5 x 106 lymphocytes in VBS was added to Con A-exposed or unexposed monocyte monolayers in 1 ml of F-10 Ham's with 1% gelatin. The lymphocytes were allowed to settle for 1-25 hr at 370C in a humid atmosphere of 5% CO2 in air. The non-adherent cells were decanted and the monolayers washed five times with HBSS, air-dried, stained with Wright's Giemsa and examined by light microscopy. Monocytes were identified by their characteristic morphologic appearance and by the presence of greater than three intracellular latex particles. One hundred consecutive monocytes were examined and the number of lymphocytes bound determined. Addition of a-MM monocytes and monocyte-lymphocyte clusters. The effect of a-MM (Sigma Chem. Co., St. Louis, Missouri) on Con A-mediated monocyte-lymphocyte interaction was assessed at several times during the culture period. a-MM (a-MM-D mannopyranoside) (10-1M-10-3M) in HBSS was added simultaneously with Con A pulsing of monocytes, with the addition of lymphocytes to Con A-treated monocytes, and 1, 24, or 72 hr after monocyte-lymphocyte clusters had formed. After incubation of the cells with a-MM for 1 hr at 370C, the plates were washed with HBSS and monocyte-bound lymphocytes quantified as above. Binding of T versus B lymphocytes to monocytes. To assess the subclass of lymphocytes binding to the Con A-pretreated monocytes, relatively enriched populations of B or T lymphocytes were prepared (Schreiber et al., 1976). Glass adherent and non-adherent populations of cells were separated as described in the first step of lymphocyte preparation. The decanted non-adherent lymphocytes were then incubated with sheep erythrocytes to form E-rosettes. This preparation, containing rosetted and non-rosetted T lymphocytes and non-rosetted B lymphocytes, was then layered over Hypaque-Ficoll and centrifuged at 4VC at 350 g as described in monocyte preparation. Ninety per cent of the bottom layer of lymphocytes were E rosettes. The sheep erythrocytes were lysed by treating for 30 sec in distilled H20, and physiological osmolality was restored by adding 3-5% NaCl. The enriched B-cell population was obtained from the interface above the Hypaque-Ficoll and contained 50% EAC and 20% E-rosetting cells as assessed by standard techniques (Jondal et al., 1972; Mendes et al., 1972). The enriched T-cell or B-cell populations were then adjusted to 5 x 106 cells per ml VBS and layered on monocyte monolayers pretreated with 10 ,g/ml Con A as described above for the 1 hr monocyte cultures. The subclass of lymphocytes that bind to Con-A-exposed monocytes was also assessed by removal of bound lymphocytes with 0-1M a-MM immediately after they attached to monocytes. The lymphocyte suspension was then washed three times in HBSS and the percentage of E-rosetting cells determined. Lymphocyte transformation. The transformation of monocyte-associated lymphocytes was assessed morphologically and by autoradiography. Monocyte-lymphocyte clusters were prepared as described above, using 4 x 106 mononuclear cells to make the monocyte monolayers. Cells were cultured at 370C under 5% CO2 in air in RPMI-1640 (Grand Island biological, Bethesda, Maryland) containing 20% autologous serum, 100 units penicillin and 100 pg streptomycin/ml. On the 4th day, 03 puCi of 3H-thymidine (New England Nuclear) was added for 4 hr and the plates then rinsed five times with RPMI-1640 to remove non-cell associated radio-activity. Cells were fixed in 1% glutraldehyde for 1 hr at 20'C. After air-drying, plates were dipped at 50'C in Kodak NTB-3 emulsion, exposed for 1 week at 50C, developed at 16'C for 2 min in Kodak Dektol, dipped 30 sec in Kodak universal stop bath, fixed for 8 min in Kodak Fixer (CAT-1971720), and rinsed in tap water for onehalf hr. Plates were also stained with Wright's-Giemsa and standard morphological criteria of lymphocyte transformation assessed (Douglas, 1971). Addition of levamisole to monocyte cultures. Levamisole (Janssen, Inc., New Brunswick, New Jersey) was diluted in culture medium and incubated with monocyte monolayers for 1 hr prior to Con A pulsing. In some experiments, levamisole was incubated with monocyte monolayers for 42 hr prior to the addition of Con A. Tissue culture plates were washed free of levamisole before the addition of Con A by rinsing five times with HBSS. Statistics. The results are expressed as mean+ s.e.m. using Student's t-test for unpaired samples.

RESULTS

Binding of lymphocytes by untreated monocytes The addition of autologous or homologous lymphocytes to monocytes that had been cultured for 1, 42, or 72 hr did not result in cell-cell binding. Furthermore, incubation of monocytes with 10 - 2_10 - 5M levamisole did not alter the ability of these monocyte preparations to bind lymphocytes.

Monocyte-lymphocyte interaction mediated by Con A Unlike untreated monocytes, monocyte monolayers pre-incubated with Con A bound substantial numbers of subsequently added lymphocytes (Fig. 1). A maximum number of lymphocytes were bound within 1-25 hr of incubation (Fig. 2). Monocytes cultured for 42 hr prior to Con A exposure bound significantly more lymphocytes than those cultured for 1 hr prior to Con A treatment (P< .01) (Fig. 3).

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Thus fifty-three lymphocytes per 100 monocytes were bound by fresh monocyte preparations, while 146 lymphocytes 100 monocytes were bound to monolayers that had beeen cultured for 42 hr prior to Con A exposure. The increased capacity of 42-hr monocyte cultures to bind lymphocytes was due to an increase in the capacity of individual monocytes to bind lymphocytes, since equal percentages (550%) of monocytes had bound lymphocytes in both the 1-hr and the 42-hr cultures. This difference may be due to an increase of Con A receptors per monocyte or an increase in the capacity of monocyte-bound Con A to mediate monocyte-lymphocyte association. The binding of lymphocytes to Con A-sensitized monocytes was dependent upon the concentration of Con A used to sensitize the monocytes (Fig. 3). This interaction appeared specific for both lymphocytes and monocytes. Human erythrocytes, added at a concentration of 5 x 107 per ml VBS and allowed to 9

FIG. 1. Monocyte-lymphocyte interaction mediated by Con A. A monocyte and three bound lymphocytes with 'uropod' projections are shown.

50

0~~~~~

40 2

0 0

30

/

20

107 0

0

L

0-5

1.0

i15

2-0

2.5

30

Time (hr)

FIG. 2. Kinetics of monocyte-lymphocyte association. Lymphocytes were allowed to settle on monocyte monolayers for varying periods of time and the unbound lymphocytes removed by washing. Each point is the mean of 2-10 experiments.

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1601 140

i

_

120 0

100

0

-J

80

_II

60 4020 0

10

30 20 Con A (pug/mI)

40

FIG. 3. Effect of culture period on monocyte-lymphocyte association. (e-0) Monocytes cultured for 1 hr; (- - - *) monocytes cultured for 42 hr; bars show mean+ s.e.m. At Con A concentrations of 5, 10 and 20jug/ml, 42-hr monocyte cultures bound significantly more lymphocytes (P< 0.01) than 1-hr monocyte cultures.

settle onto the Con A-pretreated monocyte monolayers, did not bind to monocytes. Furthermore, polymorphonuclear leucocytes preincubated with similar concentrations of Con A (5-20 jug/ml) did not bind autologous lymphocytes. Since phagocytosis of polystyrene (latex) particles may result in the internalization of Con A receptors (Douglas, 1971), we studied whether latex particle ingestion diminished the monocyte-lymphocyte interaction following exposure to Con A. Monocytes were either pre-incubated with latex for 90 min, washed and cultured for 42 hr, or first cultured for 42 hr and then exposed to latex for 90 min. Both preparations were then exposed to Con A (10 and 20 ug/ml), washed and incubated with lymphocytes in the usual manner. There was no difference between these two monocyte preparations in the extent ot monocyte-lymphocyte association (Table 2). Inhibition of monocyte-lymphocyte interaction by a-MM ac-MM is a competitive inhibitor of Con A saccharide binding sites (Sharon & Lis, 1972). When monoTABLE 2. Effect of latex phagocytosis on Con Amediated monocyte-lymphocyte interaction

Con A (pg/ml) 10 20

Lymphocytes/monocytesx 10-2 42 hr 0 hr 94+ 16 148+ 30

100+ 12 147+ 9

Monocyte monolayers were incubated for 42 hr. Monocytes were exposed to latex particles for 1-5 hr prior to (0 hr) or following the 42-hr monocyte culture. Monocyte monolayers were then exposed to Con A for 1 hr. Lymphocytes were added and allowed to settle onto the monolayer for 1-25 hr. Similar results were observed with monocytes unexposed to latex

particles.

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cytes which had been sensitized with 10 or 20 pg/ml Con A were pre-incubated for 15 min with a-MM they lost 99+ 100 of their ability to associate with lymphocytes, and when a-MM was added to monocytes simultaneously with the addition of lymphocytes, they lost 81+ 18% of their ability to associate. In addition, a-MM caused the release of 99+ 1% of lymphocytes from the monocyte surface when added after lymphocytes had been allowed to associate with monocytes and incubated for 1 hr. Thus, it appeared that the initial binding of lymphocytes to monocytes was mediated by surface-bound Con A. However, when c-MM was added 24 or 72 hr after monocyte-lymphocyte clusters had formed, a progressively decreasing effect of oa-MM was noted (Table 3). These results suggest that after 72 hr, physical interaction between monocyte and lymphocyte becomes independent of surface-bound Con A. Alterations of the monocyte surface alone are not sufficient, since Con A-sensitized monocytes cultured for 24 or 72 hr did not bind subsequently added lymphocytes in the presence of X-MM. TABLE 3. Inhibition of monocyte-lymphocyte interaction by a-MM

Time of monocyte-lymphocyte interaction

Concentration a-MM Qi M 0-01 M 0-001 M

I hr

24 hr

72 hr

97+2* 89+ 5 0+1

55+3 14+ 6 0+ 1

0+2 0+2 0+3

* Percentage decrease of lymphocytes per 100 monocytes compared to clusters not treated with GRIMM. The mean+ s.e.m. of triplicate experiments is shown.

Preferential binding of T l~mphocytes Monocyte monolayers that were pretreated with 10 pg/ml Con A bound 22+3 lymphocytes per 100 monocytes from T-cell enriched populations vs 8+ 1 lymphocytes per 100 monocytes from the Blymphocyte-enriched populations. A mixed population, as obtained for other experiments, but then recentrifuged on Hypaque-Ficoll and treated in the same manner as in the preparation of the T-cell population bound 18+2 lymphocytes per 100 monocytes. Lymphocyte populations consisting of 5500 T lymphocytes (E-rosetting cells) and 25% B lymphocytes (EAC3-rosetting cells) were added to washed monocyte-monolayers pre-incubated with Con A and allowed to attach to monocytes as above. When these lymphocytes were displaced from their monocyte attachment site by a-MM, 85% of these lymphocytes so removed formed E-rosettes.

Transformation of monocyte-associated lymphocytes Lymphocytes attached to Con A-pulsed monocytes, were large, exhibited a loose chromatin pattern and contained nucleoli (Fig. 4). They were transformed by usual morphologic criteria. When examined by means of radioautography with 3H-thymidine, 29-5 +4-30 _ of the lymphocytes bound to monocytes contained large numbers of grains, while only 10-5+3-6.60, of lymphocytes that were not associated with monocytes contained grains (P< -005) (Fig. 5). Some of the free lymphocytes may have been attached to long monocyte cytoplasmic projections, which could be seen only on phase contrast microscopy. Only 1% of lymphocytes that were cultured for 4 days in tissue culture plates that were pretreated with Con A but did not contain monocytes showed 3H-thymidine uptake.

E/Jest of levamisole on monocyte-ly1mphocy1te interaction Monocytes (1 hr cultures) exposed to levamisole before incubation with Con A bound more lymphocytes than those which were not exposed to levamisole (Fig. 6). Enhancement of binding was observed over a range of Con A concentrations (5-20 pg/ml) and was significant (P< *001) at 5-10 pg/ml Con A.

2. W. Kazura et al.

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14

I FIG. 4. Transformation of monocyte-associated lymphocytes. Two of the four lymphocytes attached to the latex-containing monocytes are large, have loose chromatin and contain nucleoli. Culture period was 96 hr. Wright's-Giemsa stain. FIG. 5. Autoradiography of monocyte-associated lymphocytes; 96-hr cultures were pulsed with 3H-thymidine for 4 hr.

40

50 0

40

"In

30

.if

20 10 0

Con A (Hag/mI)

FIG. 6. Effect of levamisole on Con A-mediated monocyte-lymphocyte interaction. (e .... e) Monocytes exposed to 2 x 10-3 M levamisole; (I-*) monocytes exposed to buffer. Levamisole significantly (P< 0.001) facilitated monocyte-lymphocyte association when monocytes were pretreated with 5 or 10 ,pg/ml of Con A. At 20,pg/ml Con A, levamisole's effect was less striking.

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80

60

20

10 I

0

0-5 1-0 Levamisole (x 10-3 M)

2-0

FIG. 7. Dose-response effect of levamisole on Con A-mediated monocyte-lymphocyte interaction. Con A concentrations were (e ... e) 5 pg/ml and (0--) 10 pg/ml. Levamisole at 1-2 x 10- 3M significantly (P< 0.01) facilitated lymphocyte-monocyte association when monocytes were pre-treated with 5 or 10 ug/ml of Con A.

Less marked enhancement was observed in 42-hr monocyte cultures. The levamisole effect required a concentration of 1 x 10- 3M (Fig. 7). Incubation of monocytes with levamisole did not result in lymphocyte binding in the absence of Con A and did not cause monocyte red-cell association, when red cells were substituted for lymphocytes in the assay.

DISCUSSION These studies demonstrate that human monocytes pretreated with the lectin Con A bind autologous lymphocytes in a dose-dependent manner, that T lymphocytes are preferentially bound, and that levamisole facilitates this interaction. The physical association between monocyte and lymphocyte is of functional significance, resulting in the mitogenic transformation of bound lymphocytes in the absence of soluble Con A. Although guinea-pig macrophages and T lymphocytes have been observed to associate in the absence of lectins (Lipsky & Rosenthal, 1973), this did not occur in our study with human cells. There may be several explanations for this apparent disparity other than an intrinsic difference between guinea-pig and human cells. First, peripheral blood monocytes are not mature cells in the sense that they have not undergone the alterations associated with tissue localization (Von Furth, 1970) and thus are not completely analogous to guinea-pig peritoneal macrophages. However, prolonged incubation of monocyte nonolayers, which led to the development of cells morphologically similar to macrophages, did not iesult in the binding of subsequently added lymphocytes. Second, our assay system depended on the settling of lymphocytes on to monocytes, unlike the rocking utilized to effect guinea-pig macrophagelymphocyte interaction (Lipsky & Rosenthal, 1973; Lipsky & Rosenthal, 1975). Cell-cell interaction mediated by Con A has been described for several cell types (Sharon & Lis, 1972; Sharon & Lis, 1975). The ability of Con A to mediate this interaction has generally been ascribed to the agglutinating property of surface-bound Con A. The initial interaction between human monocyte and lymphocyte is probably largely related to this effect, since it is markedly inhibited by a-MM. Nevertheless, the physical interaction between lymphocyte and monocyte may be unique in certain respects. The binding of lymphocytes to monocytes pre-incubated with Con A is relatively cell specific. Erythrocytes do not bind to similarly treated monocytes (although Con A-coated erythrocytes do bind to untreated monocytes (Guerry et al., 1976)) and lymphocytes do not bind to Con A-pulsed granulocytes. This preferential binding of lymphocytes to monocytes may, in part, be explained by the markedly different number and mobility of Con A receptors on lymphocytes (Sharon & Lis, 1975).

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The failure of ac-MM to displace lymphocyte-monocyte clusters after 72 hr (Table 3) suggests a lymphocyte-monocyte surface interaction independent of surface-bound Con A. This further suggests that a cell surface alteration(s) may occur during the process of monocyte-dependent lymphocyte activation. Physical interaction between monocyte and lymphocyte has been observed with neuraminidase and galactosidase treatment (Greineder & Rosenthal, 1975). These agents affect the cell surface glycoproteins of monocytes. Whether a similar cell surface alteration is involved in lymphocyte-monocyte interaction initiated by Con A is unknown. Direct monocyte- or macrophage-lymphocyte association has been observed in similar systems. Cline & Swett observed that human monocytes briefly pulsed with antigen bind autologous lymphocytes (Cline & Swett, 1968). Human monocytes and lymphocytes cultured in the presence of phytohaemagglutinin (PHA) also became associated (Epstein, Cline & Merigan, 1971). However, in our study such lymphocyte-monocyte association was observed when the monocytes alone were exposed to Con A prior to incubation with lymphocytes. Lymphocyte-macrophage association has also been observed both in antigen-dependent and antigen-indeprendent murine systems (Lipsky & Rosenthal, 1973; Lipsky & Rosenthal, 1975; Nielsen et al., 1974; Werdelin, Braendstrup & Pedersen, 1974). The light microscopic appearance in these various studies of cell-cell interaction is similar to that which we observed. Distortion of the lymphocyte surface at the point of macrophage contact, with 'uropod' formation has been noted (Cline & Swett, 1968; Nielsen et al., 1974). Similar morphologic changes have been observed in association with lymphocyte transformation; however, their precise functional significance is unknown (Nielsen et al., 1974). Although human monocytes differ from murine peritoneal macrophages in several respects (Rosenstreich, Farrar & Dougherty, 1976; Von Furth, 1970), there is evidence which suggests that both cells play an important role in the activation of T lymphocytes by mitogens. Human monocytes increase T-cell blastogenic response to Con A and PHA (Hedfors et al., 1975; Lohrmann et al., 1974) and Rosenstreich et al. (1976) demonstrated that a purified guinea-pig T-cell population proliferates in response to PHA or Con A only when reconstituted with macrophages. Two factors appear to play a role in monocyte or macrophage mediation of lymphocyte transformation: soluble products and cell-cell contact. Gery & Waksman (1972) described a lymphocyte activating factor (LAF) secreted by human monocytes and Rosenstreich et al. (1976), using a double-chambered vessel, confirmed the presence of a soluble factor in guinea-pig macrophage-lymphocyte interaction (Rosenstreich et al., 1976). However, optimal reconstitution of lymphocyte transformation in the latter study required the presence of macrophages within the lymphocyte chamber, and it was suggested that a second factor, cell-cell contact, may be required. Our results indicate that Con A-pulsed monocytes preferentially bind E-rosetting (T) cells. The transformation of lymphocytes in direct contact with Con A-pulsed monocytes (Figs 4 and 5) and infrequent transformation of lymphocytes not in direct contact with monocytes, suggests that cell-cell contact is in fact necessary for optimal human T-lymphocyte transformation. Our data provide further evidence that levamisole alters human monocyte function (Pike & Snyderman, 1976; Schreiber & Parsons, 1977). Levamisole increases either the number or the affinity of human monocyte IgG receptors (Schreiber & Parsons, 1977) and our studies show that levamisole also increases the capacity of the Con A-pretreated human monocyte to bind lymphocytes. This effect requires only a 1 hr exposure of monocytes to levamisole. Whitcomb, Merluzzi & Cooperband (1976) observed that levamisole potentiates lymphokine production from lymphocyte cultures exposed to Con A. Since monocytes may be necessary for the production of lymphokines (Epstein et al., 1971), augmented monocytelymphocyte interaction effected by levamisole may explain this observation. Levamisole has also been reported to restore defective cellular immunity in patients with neoplasia (Levo, Rotter & Ramot, 1975; Tripodi, Parks & Brugmans, 1974). In vitro studies, however, are inconclusive. Copeland, Stewart & Harris (1974) did not find an effect of levamisole on mitogen-induced transformation, but Lichtenfeld et al. (1976) did observe that levamisole increases the proliferative response to mitogens. Our data suggest that this might be due to an effect of levamisole on the human monocyte. The concentrations of levamisole employed in our study are similar to those employed by others in vitro (Anderson, Glover & Koornhoff, 1976; Pike & Snyderman, 1976; Levo et al., 1975). Only

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limited data is available on levamisole plasma levels in vivo. When measured in vivo levels have been lower (10- 6M) than concentrations employed in vitro (Symoens & Rosenthal, 1977). Nevertheless, an effect of levamisole on monocyte binding and/or presentation of antigen to the lymphocyte surface might explain, in part, levamisole's facilitation of delayed hypersensitivity in man. This study was supported by CA-15236 and HL-15061 and N.C.I. Fellowships CA-05851 (to J.K.) and CA-03150 (to D.G.), by N.I.H. Training Grant HL-06019, and by the Medical Research Service of the V.A. W.N. is a Fellow of the American Cancer Society. A.S. is a Scholar of the Leukemia Society of America. REFERENCES ANDERSON, R., GLOVER, A. & KOORNHOFF, H.J. (1976) In vitro stimulation of neutrophile mobility by levamisole. ]. Immunol. 117, 428. BENNETT, W.E. & COHN, Z.A. (1966) The isolation and selected properties of blood monocytes. ]. exp. Med. 123,

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Human monocyte-lymphocyte interaction and its enhancement by levamisole.

Clin. exp. Immunol. (1979) 35, 258-268. Human monocyte-lymphocyte interaction and its enhancement by levamisole I. W. KAZURA, W. NEGENDANK, D. GUERRY...
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