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 5 25 F 4 20 - 0 X 3 0 x '5f 0 -3 L'1 1~ 2 0 l0o Ei E J :§ 5 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.