Peritoneal Cells

Immunology 1976 30 741

Two distinct lymphocyte-stimulating soluble factors (LAF) released from murine peritoneal cells

I. THE CELLULAR SOURCE AND THE EFFECT OF cGMP ON THEIR RELEASE

T. DIA MANTSTEIN & A. UL MER Immunological Research Unit, Klinikum Steglitz Freie Universitdt, Berlin, Germany

Received 26 September 1975; acceptedfor publication 30 October 1975

Summary. Culture fluids of murine peritoneal cells similar lymphocyte (predominantly thymocyte)- contain two distinct, non-dialysable principles activating factor(LAF) and lymphocyteproliferation- (LAF) with thymocyte proliferation-stimulating inhibiting factor (LIF) can be obtained by incubat- properties. One of them is elaborated from phago- ing normal peritoneal mouse cells (rich in non- cytic cells (presumably macrophages), the other is stimulated macrophages) in the presence of cyclic- released by non-phagocytic cells (lymphocytes). 3',5'-guanosine monophosphate (cGMP). This paper cGMP added exogenously stimulates the production reports in detail on this phenomenon with respect and/or release of LAF from phagocytic cells, but not to the specificity of cGMP as an inducer for LAF from non-phagocytic cells. The phagocytic cells (but release and the identity of the cells releasing LAF in not the lymphocytes) require intact RNA and pro- presence and absence ofcGMP. tein synthesis for LAF release. The action of LAF differs from those of cGMP itself by being non- dialysable, and unable to prevent the inhibitory MATERIALS AND METHODS action of cAMP on mitogen-stimulated lymphocyte proliferation. Culture fluids BALB/c mice 8-12 weeks old were killed and their INTRODUCTION peritoneal cells (containing up to 85 per cent macro- Factor(s) that induce, potentiate or inhibit lympho- phages) were harvested 5 min after intraperitoneal cyte proliferation have been found in culture fluids injection of 6 ml of RPMI-1640 medium (Gibco/ derived from lymphoid cells of various sources Biocult, Glasgow) supplemented with 2 mmol including proteose peptone-induced peritoneal exu- L-glutamine 100 u of penicillin and 100 ,ug of strep- date cells incubated in vitro with or without endo- tomycin per ml. The cells were centrifuged off (250 g, toxin (Gery, Gershon and Waksman, 1972; Gery and 10 min), washed and suspended at a density of 2 x 106 Waksman, 1972; Calderon, Williams and Unanue, per ml in the same medium. One millilitre of cell sus- 1974; Calderon and Unanue, 1975). Recently, we pension containing the test compounds cGMP, reported (Diamantstein and Ulmer, 1975a) that a guanosine-5'-monophosphate (GMP), guanosine-5'- Correspondence: T. Diamantstein, Klinikum Steglitz diphosphate (GDP) or guanosine-5'-triphosphate Freie Universittt, 1 Berlin 45, Hindenburgdamm 30, (GTP) (purchased from Boehringer, Mannheim) Germany. were incubated in 16 x 124 mm disposable plastic 741 742 T. Diamantstein & A. Ulmer tubes (Falcon Plastics, Los Angeles, California) at cultures expressed as counts per minute (c.p.m.) or as 370 in 5 per cent CO2 atmosphere for various times stimulation indices (SI) = the ratio of c.p.m. of (usually for 24 or 48 h). In some experiments 5 pg/ml experimental cultures to control cultures not contain- actinomycin D (Boehringer, Mannheim) or 200 ing CF derived from PC cultures. In PHA-stimulated pg/ml cycloheximide (Serva, Heidelberg) were added thymocyte cultures the results are expressed as to the cultures. Culture fluids (CF) were harvested, c.p.m. and as potentiating indices (PI) = the ratio of sterilized by Millipore filtration (crude CF) or dia- c.p.m. of PHA- and CF-containing cultures to con- lysed for 2 days against RPMI-1640 medium (with trol cultures not containing CF. two changes of the dialysate per day) before Milli- pore filtration (dialysed CF). To obtain peritoneal cells (PC) depleted of phago- RESULTS cytic cells, 2 x 107 peritoneal cells suspended in 1 ml of RPMI-1640 medium supplemented with 20 per Studies on the specificity of the effect of cGMP and cent foetal calf serum (Gibco/Biocult, Glasgow) were the lack of serum requirement for LAF release mixed with 3 ml of a suspension of carbonyl iron powder (lymphocyte-separating reagent, LSR, Tech- PC were cultured in presence or in absence of nicon Instruments, Tarytown, New York). The sus- human serum with various guanosine nucleotides. pensions were incubated in 60 x 15 mm tissue culture Plastics) at 370 for 30 min in a 5 per dishes (Falcon Table 1. The effect ofvarious guanosine nucleo- cent CO2 atmosphere on a rocker platform (Bellco, tides on LAF release Vineland, New Jersey) at 10 oscillations per min. The phagocytic cells containing the iron were removed by LAF activity placing the dishes on a permanent magnet (Eclipse, Culture conditions Sheffield) for 5 min. This procedure was repeated for LAF production c.p.m.fCulture SI twice. The non-adherent lymphocyte-rich cell popu- PC+GMP (5 mM) 25±2 1-4 lation (up to 90 per cent lymphocytes) was then used. PC+GDP (2 mM) 118±12 6-6 PC+GTP (5 mM) 20±1 1-4 Test systems for lymphocyte activating factor (LAF) PC+c-GMP (5 mM) 82±5 4-6 content of the culture fields PC 17±2 09 A thymocyte suspension derived from BALB/c mice CF derived from 24-h peritoneal cell (PC) was by conventional procedures as des- prepared cultures were added at a concentration of 10 cribed earlier (Diamantstein, Vogt, Ruhl and per cent to thymocyte cultures. The results are Bochert, 1973) and cultured in RPMI-1640 medium, expressed as mean c.p.m. per triplicate cul- as described above, but supplemented with 5 per cent tures+ s.e.m., and as stimulation indices (SI). heat-inactivated fresh human serum. The cells in a For experimental details see the Materials and Methods section. total volume of 0 15 ml (2 x 106 cells per ml) were cultured with or without PHA (Phytohemagglutinin- P, Difco, Detroit, 10 pg/ml) in Microtitre plates Table 2. Lack of serum requirement for stimu- M24AR, system Cooke (C. A. Greiner, Nuirtingen) lation of LAF release by cGMP in a 5 per cent CO2 atmosphere at 370 for 48 h. Ten microlitres of [3H]thymidine (01 pCi, 2 Ci/mM) LAF activity Culture conditions (Radiochemical Centre, Amersham, Bucks), was for LAF production c.p.m./Culture SI added to the cultures for the last 4 h of incubation period. Cells were collected on glass fibre filters using PC+serum+cGMP 148±6 10 6 a Skatron multiple cell culture harvester and the in- PC+cGMP 153±34 10.9 1 2 of into the nuclear DNA PC+ serum 17±1 corporation [3H]thymidine PC 14±2 10 was then determined as described previously (Diam- For the antstein and Ulmer, 1975b). testing LAF, Peritoneal cells (PC) were cultured with or thymocytes or PHA-stimulated thymocytes were without 5 per centheat-inactivated fresh human cultured in presence of various concentrations of CF serum in presence or absence of 5 mm cGMP of various origin. The results are means of triplicate for 24 h. For experimental details see Table 1. LAFfrom peritoneal cells. I. 743

Twenty-four hours later the culture fluids were h in medium containing various concentrations of collected and tested for LAF activity on thymocytes. crude or dialysed CF. A typical experiment is Results in Table 1 show that apart from cGMP, shown in Fig. 1. As measured by thymidine uptake, GDP stimulated the production and/or release of both the crude, as well as the dialysed CF derived LAF into the CF. As shown in Table 2, serum was from cGMP-treated PC cultures contained LAF. In not required to obtain a LAF-containing supernatant. this experiment maximum stimulation was observed Therefore, further experiments were performed with at a CF concentration of 30 per cent. However, a high PC cultured in medium without serum. variation in the LAF content was observed using CF derived from various cGMP-treated PC cultures. In thirteen different preparations (data not given here) The dose-response of thymocytes to culture fluids of the maximum stimulation of thymocytes occurred at cGMP-stimulated peritoneal cells a dialysed CF concentration ranging from 10 to 50 indices of 3-30. At a con- 48 per cent with stimulation Culture fluids were obtained by incubating PC for the CF was in absence of cGMP in serum- centration of 50 per cent crude markedly h in the presence or than the CF indicating the free medium. Aliquots of crude culture fluids (crude less effective dialysed of a dialysable inhibitory principle des- were against culture medium presence CF) extensively dialysed et al., 1974). No sig- (dialysed CF). Thymocytes were then cultured for 48 cribed previously (Calderon nificant amount of LAF could be detected in either

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0 I I 0 1 2 3 5 10 20 30 50 2 3 5 10 20 30 50 CF (percent) CF (per cent) Figure 1. Dose-response of thymocytes to LAF of various Figure 2. Dose-response of PHA-stimulated thymocytes to sources. Thymocytes (2 x 106 per ml) were cultured in medium LAF of various sources. Thymocytes (2 x 106/ml) were cul- containing various concentrations of crude or dialysed CF tured with 10 jug/ml PHA in medium containing various con- derived from peritoneal cells incubated for 48 h in presence or centrations of crude or dialysed CF. For details see Fig. 1. (0) in absence of 5 mm cGMP. The thymidine incorporation was PHA-stimulated thymocytes cultured in presence of crude determined in 48-h cultures. Each value represents the mean CF derived from peritoneal cells cultured with cGMP; (A) c.p.m. detected in triplicate cultures, s.e.m. < 10 per cent. (0) PHA-stimulated thymocytes cultured in presence of dialysed Thymocytes cultured in the presence of crude CF derived CF derived from peritoneal cells cultured with cGMP; (0) from PC cultured with cGMP; (A) thymocytes cultured in PHA-stimulated thymocytes cultured in presence of crude the presence of dialysed CF derived from PC cultured with CF derived from peritoneal cells cultured without test sub- cG MP; (0) thymocytes cultured in the presence of crude CF stances; (A) PHA-stimulated thymocytes cultured in presence derived from PC cultured without test substances; (A) thymo- of dialysed CF derived from peritoneal cells cultured without cytes cultured in presence of dialysed CF derived from PC test substances. PHA-stimulated thymocytes cultured without cultured without test substances. Thymocytes cultured with- CF: 142+11 c.p.m./culture. Thymocytes cultured without out CF: 23+ 1 c.p.m./culture. PHA and without CF: 23+ 1 c.p.m./culture.

K 744 T. Diamantstein & A. Ulmer

2 6 12 24 48 72 2 6 12 24 48 72 Hours Figure 3. The kinetics of LAF release. Peritoneal cells (2 x 106/ml) were incubated in presence or absence of 5 mm cGMP. Culture fluids were collected at various times, dialysed and tested at concentration of (a) 10 per cent or (b) 50 per cent for LAF activity on PHA-stimulated thymocyte cultures. The results are expressed as potentiation indices. Each value represents the mean of triplicate cultures; s.e.m. < 10 per cent. (0) PHA-stimulated thymocytes cultured in presence of dialysed CF derived from peritoneal cells cultured with cGMP; (0) PHA-stimulated thymocytes cultured in presence of dialysed CF derived from peritoneal cells cultured without test substances. crude or dialysed CF derived from PC cultured in Inhibition of stimulatory effect of cGMP on LAF the absence ofcGMP. production by cycloheximide and by actinomycin D may induce simply release fluids To show whether cGMP The potentiation of the PHA response by culture LAF from PC or whether pro- PC of already performed of cGMP stimulated and non-stimulated tein and/or RNA synthesis is required for this effect, Crude or dialysed culture fluids derived from either PC were cultured with or without cGMP in presence cGMP-treated or control PC cultures were added at or in absence ofeither cycloheximide or actinomycin various concentrations to thymocyte cultures in pre- D. The culture fluids were collected, dialysed and sence of a mitogenic dose of PHA. Although all the culture fluids tested potentiated the response of thy- Table 3. The effect of actinomycin D and cycloheximide on mocytes to PHA, the degree of their stimulatory stimulation of LAF release by cGMP activity differed markedly (Fig. 2). Culture fluids derived from cGMP-treated PC cultures contained LAF activity conditions a LAF activity than culture fluids of non- Culture higher for LAF production c.p.m./Culture PI stimulated PC. Dialysis enhanced markedly the stimulatory activity of the culture fluids. PC+cGMP (5 mM) 783+45 6-1 PC 226+ 38 1-8 The effect of cGMP on the kinetics of the appearance PC+c-GMP (5 mM)+cycloheximide (200 pg/ml) 284+ 7 2-2 of LAF in the culture fluids of PC PC+ cycloheximide (200 pug/ml) 195+2 1P5 PC+ cGMP (5 mM)+ actinomycin with or without cGMP for various PC were cultured D (5pgfml) 134±15 1.1 periods. The culture fluids were then collected and PC+ actinomycin D (5 4uglml) 174+6 1-4 dialysed. The effect of dialysed CF (added at 10 or at 50 per cent concentration to PHA-stimulated thymo- Peritoneal cells (PC) were cultured for 24 h under condi- cyte cultures) on the thymidine incorporation in tions given in the table. The dialysed CF were tested at a con- was detected (Fig. 3a, b). The LAF centration of 10 per cent on PHA-stimulated thymocyte thymocytes detected in cGMP-treated PC cultures cultures. The results are expressed as mean c.p.m. activity obtained from triplicate cultures+ s.e.m. and as potentiation indices (PI). was higher than those of control culture fluids at any Thymocyte cultures with PHA in absence of CF gave 128 + 3 time tested. Moreover, cGMP treatment increased c.p.m., controls (thymocyte in absence of PHA and of CF) the LAF concentration in the fluids of 6-h cultures. 14± 2 c.p.m. LAFfrom peritoneal cells. I. 745 tested for LAF activity on PHA-stimulated thymo- Table 4. The effect of LAF on thymocyte mitosis cytes. The results of these experiments (Table 3) show that both intact protein as well as RNA synthesis Cell mitosis Thymidine (percentage incorporation are required for stimulation of LAF release and Mitogen metaphases) (c.p.m./culture) production by cGMP. However, the cGMP independent (background) LAF activity was not affected PHA+LAF 3.9 2574+ 134 PHA 04 142+ 11 either by cycloheximide or by actinomycin D. Nil <01 18+2

Effect of LAF on the percentage of cells in colcemide 2 x 106 thymocytes were incubated for 72 h in 1 ml of cul- metaphase ture medium with PHA (10 pg/ml) or with PHA and LAF (20 per cent dialysed CF derived from peritoneal cells incubated Culture fluids of cGMP stimulated PC (six cultures) with 5 mm cGMP for 48 h). 0-1 pug of colcemid (Geigy, Basel) were collected and pooled. One millilitre of thymo- was added to the cultures for the last 6 h of the incubation cyte suspension (1 x 106 cells) was then cultured period. The cells were swollen in hypotonic solution (75 mm with PHA in the presence of 20 per cent dialysed KCI), fixed with acetic acid: methanol (1: 3), stained by Giemsa and examined for mitotic spreads. The results are expressed CF and the number of cells in colcemide metaphase as mean percentage of nuclei in metaphase of triplicate cul- determined in 72-h cultures. Aliquots of cells were tures. The thymidine incorporation into thymocytes was cultured under identical conditions in the usual way determined in parallel 48-h cultures as described in the and the thymidine uptake was determined in 48-h Materials and Methods section. In this case, the results are + s.e.m. cultures. The effect of LAF on cell mitosis (Table 4) expressed as c.p.m. of triplicate cultures. parallels the results obtained by detecting [3H]- thymidine uptake by thecells. Theresults demonstrate production only when normal peritoneal cells were that LAF, indeed, affects cell mitosis and not only used (containing the phagocytic cells). An unexpect- alters the thymidine uptake by the cells. ed cGMP-independent release of LAF, apparently not from phagocytic cells, could be obtained incubat- The cellular source of cGMP-stimulated and ing purified lymphocyte derived from the peritoneal background LAF production cavity (Table 5). PC or PC depleted of phagocytic cells were cultured Difference between LAF and cGMP activity either in presence or in absence of cGMP. Twenty- four hours later, the culture fluids were collected, Spleen cells were cultured with mitogenic doses of dialysed and tested for LAF content on PHA- LPS, PHA, cGMP or LAF in presence or absence stimulated thymocytes. LAF was detectableinculture of cyclic 3',5'-adenosine monophosphate (cAMP). fluids derived as well from normal PC as from PC LPS (lipopolysaccharide of E. coli 055:B5, Difco; depleted of phagocytic cells. cGMP stimulated LAF Detroit) or PHA-treated spleen cells were cultured

Table 5. The cellular source of LAF

Ten per cent CF in the Twenty per cent CF in the medium LAF activity medium LAF activity Culture conditions for LAF production c.p.m./Culture PI c.p.m./Culture Pi PC 185+ 11 2-2 381+43 4-4 PC+cGMP 621+30 7-2 928+69 10-7 Non-phagocytic PC 482+ 32 5 6 852+ 159 9 9 Non-phagocytic PC+ cGMP 601+ 35 7*0 1012+ 53 11-8

Peritoneal cells (14 per cent lymphocytes, 84 per cent macrophages, 2 per cent granulocytes) or peritoneal cells depleted of phagocytic cells (88 per cent lymphocytes, 10 per cent macrophages, 2 per cent granulocytes) were cultured for 24 h in presence of absence of cGMP (5 mM). For testing LAF activity, PHA-stimulated thymocytes were cultured with dialysed CF at concentrations of 10 and 20 per cent respectively. The results are expressed as mean c.p.m. detected in triplicate cultures+ s.e.m. and as potentiating indices (PI). 746 T. Diamantstein & A. Ulmer Table 6. The lack of capacity of LAF to Unanue, 1975) with lymphocyte proliferation-in- antagonize the inhibitory action ofc-AMP hibiting properties (LIF). The cellular source of LAF on mitogen-stimulated spleen cells and of LIF has been claimed to be adherent cells Mitogen c.p.m./Culture (Gery and Waksman, 1972; Calderon et al., 1975), presumably macrophages. Recently, we reported PHA 2234+ 34 (Diamantstein and Ulmer, 1975a) that cGMP induces LPS 1886+ 16 release and/or production of LAF by non-stimulated cGMP 552+ 9 murine peritoneal cells. The present work is con- LAF 354+ 18 PHA+cAMP 33+ 3 cerned with the cellular source of LAF and some of LPS+cAMP 240+ 2 its properties. The main finding reported here is that cGMP+ cAMP 254+ 7 two different types of LAF can be distinguished LAF+cAMP 59+ 7 in dialysed culture fluids of murine peritoneal cells. PHA+cAMP+cGMP 784+ 31 and can be also LPS+cAMP+cGMP 1258+ 39 Both principles are non-dialysable PHA+cAMP+LAF 54+ 2 detected in peritoneal cell suspensions cultured in LPS+ cAMP+ LAF 296+ 8 the absence of serum. LAF elaboration is not related Nil 91+ 5 with a mitotic response of the donor cell since it has been observed in culture fluids of endotoxin (Gery BALB/c spleen cells (2 x 106 per ml) and Waksman, 1972) as well as cGMP treated cells were cultured for 48 h (under conditions and Ulmer, 1975a) unable to respond described in the Materials and Methods (Diamantstein section for thymocytes) with PHA (10 mitotically to these compounds. jug/ml), LPS (50,pg/ml), cGMP (5 mM), or As shown in experiments with colcemide, LAF LAF (50 per cent dialysed, CF-derived induce, indeed, cell mitosis and not only increase in from peritoneal cells cultured for 48 h in thymidine uptake. The two kinds of LAF differ in the presence of 5 mm cGMP), in presence or absence of cAMP (1 mM). Other spleen many important aspects. At first, the production/or cells stimulated with PHA or LPS were release ofone ofthe LAF is dependent on the presence incubated in presence of cAMP and with of cGMP, while the other LAF is found also in peri- either cGMP or LAF. The results are ex- toneal cell cultures in absence of this compound. The pressed as mean c.p.m. detected in tripli- cGMP-dependent LAF is produced and/or released cate cultures ± s.e.m. by phagocytic cells (mostly macrophages) since re- in presence ofcAMP sufficient to inhibit themitogenic moval of phagocytic cells diminished significantly response of the cells (Table 6). We could confirm our the capacity of cGMP to enhance the elaboration of previous finding showing that cGMP can abolish this factor. The marginally enhanced elaboration of the inhibitory action of cAMP on either LPS- or LAF in presence of cGMP by lymphocyte prepara- PHA-stimulated lymphocytes (Diamantstein and Ul- tions of peritoneal origin depleted of phagocytic mer, 1975b). However, LAF failed to do so. cells might be due to contamination of the lymphocyte preparation used with phagocytic cells (see legend to Table 5). Moreover preliminary experi- DISCUSSION ments showed that cGMP even inhibited the elabo- Lymphocyte-activating factor(s) (LAF) have been ration of LAF by lymphocytes of splenic or thymic detected in culture fluids of cells derived from various origin. From this point of view, the cGMP-depen- lymphoid organs and ofhuman leucocytes stimulated dent LAF resembles the LAF described by others in vitro byendotoxin or bylectins (GeryandWaksman, (Gery and Handschuhmacher, 1974; Calderon and 1972; Gery and Handschuhmacher, 1974). Murine Unanue, 1975). The cGMP-independent LAF is peritoneal exudate cells (Calderon and Unanue, found in culture fluids of non-adherent peritoneal 1975) released a similar active principle into the cells (mostly lymphocytes). Moreover, the cGMP- culture fluids. The active principles have been re- dependent LAF is probably not found or, at least, ported to be non-dialysable (Calderon and Unanue, is not released by the cells in its final active form 1975; Calderon, Kiely, Lefko and Unanue, 1975; because addition of compounds inhibiting RNA Gery and Handschuhmacher, 1974) and masked (actinomycin D) or protein synthesis (cycloheximide) sometimes by a dialysable low molecular weight prevent the appearance of LAF in the culture fluid. material (Calderon et al., 1974; Calderon and On the other hand, the LAF production/release LAFfrom peritoneal cells. L 747

from the non-adherent peritoneal cell population is of T cells (Calderon et al., 1975), indicate a possible not affected by these compounds. The two types of role for LAF in the initiation ofthe immune response. LAF differ also in their spectrum of activity; the cGMP-dependent LAF acts on PHA-stimulated as well as on non-stimulated thymocytes, while the other ACKNOWLEDGMENT LAF seems to act only on PHA-stimulated thymocytes. This work was supported by the Deutsche The presence of LAF in culture fluids of non- Forschungsgemeinschaft. adherent cells has been reported by others (Di Sabato, Chen and Erickson, 1975). Our results confirm these findings by using non-adherent peritoneal cells and extend them by showing the cGMP independence REFERENCES of this factor. case was CALDERON J., KIELY J.A., LEFKO J.L. & UNANUE E.R. (1975) In view of the fact that in our LAF re- The modulation of lymphocyte function by molecules leased from macrophages only in presence of cGMP, secreted by macrophages. J. exp. Med. 142, 151. it is necessary to comment in what respect this factor CALDERON J. & UNANUE E.R. (1975) Two biological activities is different from cGMP which has also stimulating regulating cell proliferation found in cultures ofperitoneal properties (Weinstein, Chambers, Bourne and Mel- exudate cells. Nature (Lond.), 253, 359. CALDERON J., WILLIAMS R.T. & UNANUE E.R. (1974) An mon, 1974; Watson, 1975; Diamantstein and Ulmer, inhibitor of cell proliferation released by cultures of 1975b). In our opinion, LAF is not identical with macrophages. Proc. nat. Acad. Sci. (Wash.), 71,4273. cGMP because, in contrast to cGMP, LAF is not DIAMANTSTEIN T. & ULMER A. (1975a) Stimulation by cyclic dialysable and, in contrast to cGMP (Diamantstein GMP of lymphocytes mediated by soluble factor released the from adherent cells. Nature (Lond.), 256, 418. and Ulmer, 1975b) it does not prevent inhibitory DIAMANTSTEIN T. & ULMER A. (1975b) The antagonistic effect of cAMP on the stimulatory activity of either action of cyclic GMP and cyclic AMP on proliferation of LPS or PHA. B and T lymphocytes. Immunology, 28,113. Concerning the specificity of the cGMP effect on DIAMANTSTEIN T. & ULMER A (1975c) Regulation of DNA LAF elaboration, it should be mentioned that GDP synthesis by guanosine-5'-diphosphate, cyclic guanosine-3',5'-monophosphate, and cyclic adenosine-3',5- also had the same effect. Other guanosine nucleotides monophosphate in mouse lymphoid cells. Exp. Cell. Res. tested as well as 2',3'-cGMP and cAMP (Diamant- 93, 309. stein and Ulmer, 1975a) were not effective. As DIAMANTSTEIN T., VOGT W., RUHL H. & BOCHERT G. (1973) shown previously (Diamantstein and Ulmer, 1975c), Stimulation of DNA synthesis in mouse lymphoid cells by GDP proliferation polyanions in vitro. Europ. J. Immunol. 3,488. enhanced of cells derived from Di SABATO G., CHEN D.M. & ERICKSON J.W. (1975) Pro- various lymphoid organs, in a similar manner to duction by murine spleen cells of an activity stimulating cGMP. It might be that, at least part of the effect the PHA-responsiveness of thymus lymphocytes. Cell. of both GDP and cGMP is due to release of LAF Immunol. 17,495. acting on these cells. It has been reported that certain GERY I., GERSHON R.K. & WAKSMAN B.H. (1972) Potentia- tion of the T-lymphocyte response to mitogens: I. The compounds, e.g. LPS, induce both LAF production responding cell. J. exp. Med. 136, 128. (Gery and Waksman, 1972) and increase the intra- GERY I. & WAKSMAN B.H. (1972) Potentiation of the T- cellular concentration of cGMP (Watson, 1975) lymphocyte response to mitogens. II. The cellular source in leucocytes. These facts and the results in this ofpotentiating mediator(s). J. exp. Med. 136, 143. paper showing stimulation of LAF elaboration by GERY I., HANDSCHUHMACHER R.E. (1974) Potentiation of the T-lymphocyte response to mitogens. III. Properties of the exogenously added cGMP suggested that a cGMP- mediator(s) from adherent cells. Cell. Immunol. 11, 162. dependent step may be involved in the production of WATSON J. (1975) The influence of intracellular levels of this factor. The biological significance of various cyclic nucleotides on cell proliferation and the induction kinds ofLAF is not yet clear. Recent reports showing ofantibody synthesis. J. exp. Med. 141, 97. WEINSTEIN Y., CHAMBERS D.A., BOURNE H.R. & MELMON that the immune response in vitro is enhanced under K.L. (1974) Cyclic GMP stimulates lymphocyte nucleic limiting conditions by LAF, probably via activation acid synthesis. Nature (Lond.), 251, 352.