Immunology 1977 32 215

Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs J. B. LEIPER * & J. B. SOLO MON Immunology Unit, Department ofBacteriology, University ofAberdeen, Aberdeen

Received 29 January 1976; accepted for publication 5 August 1976

The onset and maturation of mitogenic responsiveness in the guinea-pig and human foetus is compared by age-equivalence.

Summary. Pre-culture of spleen cells for 24 h before addition of activator greatly improved the mitogenic response to phytohaemagglutinin (PHA) and sheep erythrocytes (SRBC). Spleen cells were separated into plastic-adherent and non-adherent populations and in some experiments purified further to 'macrophages' and 'lymphocytes' respectively. A critical mixture of lymphocytes and macrophages (10:1) gave twice the mitogenic response of lymphocytes alone to PHA and SRBC in cultures of foetal, juvenile and adult guinea-pig spleen cells. Vigorous mitogenic responses to PHA and SRBC were found at 36 days of gestation. The mitogenic response increased from 46-56 days of gestation to above adult level. Lipopolysaccharide (LPS) obtained by the Westphal method was a superior mitogen to LPS extracted by the Boivin technique. Plaqueforming cells (PFC) could not be induced in vitro at any age even with the addition of LPS. Evidence was obtained for mitogenic and blastogenic lymphokines produced by lymphocytes activated with PHA or SRBC (but not LPS). These lymphokines were produced by activated splenic lymphocytes of a 40-dayold foetus; older animals showed no evidence for a quantitative increase in lymphokine production.

INTRODUCTION The onset of responsiveness to phytohaemagglutinin (PHA) in cultures of foetal guinea-pig thymus cells occurs as soon as sufficient thymus cells can be obtained for a culture (at 35 days post-coitus) (Reid, Leiper, Inglis & Solomon, 1973). No age-related differences in the mitogenic response were observed. We have now adopted a more sensitive microplate culture technique to obtain much greater uptake of H3-labelled thymidine in cultures of spleencellstaken from foetal guinea-pigs. Responsiveness to PHA has also been increased by 'ageing' spleen cells in culture prior to the addition of PHA (Leiper & Solomon, in preparation). The ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes in cultures of foetal guinea-pig spleen cells has been determined. These studies reveal an age-related increase in responsiveness from 36 days post-coitus to adult levels just before birth. The onset and maturation of responsiveness to PHA in foetal life is compared to previous work in mice and man by means of age-equivalence (Solomon, 1971). This increase in responsiveness to mitogens during foetal life may be due to the appearance of lympho-

* Present address: Department of Surgery, University of Aberdeen, Aberdeen AB9 2ZD. Correspondence: Dr J. B. Solomon, Immunology Unit, Bacteriology Department, University of Aberdeen, Aberdeen AB9 2ZD.



J. B. Leiper & J. B. Solomon

kines during ontogenetic development. The ability of foetal, juvenile and adult lymphocytes to produce mitogenic and blastogenic lymphokines was examined. These lymphokines appear early in foetal life (40 days of gestation) and there are no quantitative age-related changes in the extent of their production. These lymphokines are produced by lymphocytes activated with PHA or SRBC and can elicit almost as vigorous a mitogenic and blast-cell response in assay cultures as the activators themselves.


Animals Charles River, random bred, albino guinea-pigs were mated and inspected for vaginal plugs each day. Vaginal plugs give some indication of day zero postcoitus, and this was confirmed by reference to foetal body weight tables (Draper, 1920).

Cell preparation Spleens were removed aseptically, placed in sterile plastic petri dishes and any adherent tissue removed. Cell suspensions were prepared in RPM1 1640 medium with 25 mm HEPES buffer (Flow Laboratories) supplemented with penicillin (100 i.u.fml), Streptomycin (100 pg/ml) (Crystamycin, Glaxo Laboratories Ltd), glutamine (2 mM) and mycostatin (100 ,ug/ml, Squibb and Sons). The pH was adjusted to 7-2 with sodium hydroxide and membranefiltered immediately before use. Spleens were gently teased in medium (approximately 1 ml) using two sterile forceps, aspirated through 21-gauge then 25-gauge needles to disperse clumps and washed three times in medium. Viable cells were counted as previously described (Reid et al., 1973). Ageing of cell cultures Spleen cells (4 x 106) were cultured in RPMI 1640 medium with the additives described above. 5 per cent foetal calf serum (Flow Laboratories) was also added. Foetal calf serum was only used when ageing cultures. Cells were cultured in sterile petri dishes (Falcon) without activator at 370 for 24 h in 95 per cent air and 5 per cent CO2. The 24-h incubation appeared to select those cells best able to adapt to in vitro conditions.

Separation ofplastic-adherent and non-adherent cell populations Non-adherent (NA) spleen cells were separated

from the plastic-adherent (A) cells in the aged cell cultures. Dishes were gently shaken on an orbital shaker (Luckhams Ltd.) at 40 r.p.m. for 3 min and the NA cells poured off into sterile siliconized centrifuge tubes. Two further washes with warm medium removed the remaining NA cells; all NAcells were pooled. 'A' cells were gently aspirated off the bottom of the dishes with warm medium into sterile siliconized centrifuge tubes. Both cell populations were washed three times to remove foetal calf serum and counted. NA cells had a small variable number of macrophages; 'A' cells always contained more than 95 per cent macrophages. Preparation of highly purified lymphocytes and macrophages Spleen cells were incubated with 30 mg carbonyl iron, type SF (G.A.F.) with rotation at 37° for 30 min. The cell suspension was passed through the jaws of a magnet to remove the cells which had phagocytosed carbonyl iron. 'Non-phagocytic' cells were pipetted off and layered on to a Ficol-Hypaque gradient (Pharmacia/Winthrop) and spun at 1500 r.p.m. for 20 min. The lymphocyte-rich layer was removed, washed three times in medium and aged as previously described. After 24 h, the plastic-adherent and non-adherent cell populations were separated as detailed. The non-adherent population was incubated as before with 5 mg carbonyl iron and any phagocytic cells removed, the remainder of the cells were washed twice with medium. When 5000 non-adherent cells were individually examined no macrophages were observed and more than 97 per cent appeared to be typical splenic lymphocytes; this cell population is termed 'lymphocytes'. Individual examination of 5000 cells of the adherent population which had 'escaped' carbonyl iron treatment, revealed that every cell was a macrophage. Mitomycin C-treatment of macrophages and

lymphocytes Cells (3 x 106) from the purified macrophage or purified lymphocyte preparations were incubated with 40 ,g Mitomycin C (Sigma) for 30 min, washed three times with medium and adjusted to the appropriate cell concentration in 50 ,l medium. Cell culture One type of cell culture consisted of non-adherent (NA) cells (2 x 105) mixed with adherent (A) cells (2 x 104) in 0 1 ml RPMI 1640 medium with the

Mitogen response to PHA and SRBC additives previously described but without foetal calf serum. The second type of culture contained highly purified lymphocytes (2 x 105) with highly purified macrophages (2 x 104) in the same volume of medium, again without foetal calf serum. The appropriate concentration of activator in 0Q1 ml medium was added to bring the final volume in the well to 0-2 ml. Triplicate cultures of each were usually prepared. The wells were sealed using pressure sensitive film (Falcon). Cultures were incubated at 370 in 95 per cent air and 5 per cent CO2 in a humidified atmosphere. Activators Phytohaemagglutinin-P (PHA) and sheep erythrocytes (SRBC) were obtained from Wellcome Reagents Ltd. Lipopolysaccharide (LPS) from Salmonella typhosa (Boivin extraction and Westphal extraction) were obtained from Difco Laboratories and detoxified by boiling in phosphate-buffered saline (pH 7 8) for 1 h. SRBC were stored in Alsever's solution for 2 weeks at 40 in order to ensure any leucocytes were dead; when required they were washed three times in medium and counted immediately before use. PHA was stored freeze-dried in aliquots and reconstituted just before use. Optimal concentrations of PHA (02 ,pg/culture well) and SRBC (1 x 107/culture well) were used throughout these studies, except where indicated (Leiper & Solomon, in preparation).

Lymphokine production and assay Highly purified lymphocytes (2 x 105) and macrophages (2 x 104) obtained from the same spleen were cultured with or without activator (PHA, LPS or SRBC). Foetal calf serum was not used in these cultures. Supernatants were removed aseptically, pooled and centrifuged at 2400 g for 20 min to remove cells and debris. Aliquots of the supernatants were examined microscopically for evidence of cellular contamination. Aliquots (01 ml) of supernatants were then added to cultures (0-1 ml) of NA cells (2 x 105) and A cells (2 x 104) prepared from the same spleen. These assay (NA/A) cultures had been initiated 48 h earlier but no activator added. After addition of supernatants these assay cultures were maintained for a further 48 h when their mitogenic response was measured. This assayed what we term 'mitogenic lymphokine'. Blast cell counts were carried out on the same assay cultures and thus assayed 'blastogenic lymphokine'. Al-


though significant correlation between c.p.m. and percentage blast cells has been found we felt that one lymphokine may not induce both cell division and differentiation. Blast cell and macrophage counts Cells were resuspended and pipetted from individual cultures. Cell smears were made using a Cytospin Centrifuge (Shandon Southern Instruments), and routinely stained with May-Griinwald-Giemsa. Blast cells were counted as previously described (Reid et al., 1973). Two types of macrophages were seen. The majority were large, rounded cells with abundant weakly basophilic, heavily vacuolated cytoplasm and a large rounded or indented nucleus. The minority were stellate forms of macrophages whose numbers increased with prolonged culture. Cultures were also stained with Neutral Red and counted either as suspensions or as May-GrunwaldGiemsa smears. Viable macrophages typically produced a rosette of red-stained granules near the perinucleus with extensive red staining of the cytoplasm. More than 1000 nucleated cells were counted and the percentage of blast cells and macrophages determined.

Peritoneal cavity cells Cells were collected by washing the peritoneal cavity several times with warm medium. Pulsing with tritiated thymidine Tritiated thymidine (1 pCi) (specific activity 2 0 Ci/ mM; Radiochemical Centre, Amersham) in 0-1 ml medium was added to each well 1 h before termination of the cultures. This short pulse time effectively labelled the dividing cells and eliminated the possibility of infection masking the spleen cell mitogenic response. Background counts were obtained by setting up triplicate cultures of PHA, LPS or SRBC and medium without spleen cells. These background cultures were processed in the same way as the control and experimental cultures (mean background culture counts were 36-8 ± 1-6 c.p.m.). A Skatron semi-automatic cell harvester was used to harvest the water-lysed cell fragments from the wells on to glass fibre discs. These discs were dried, placed in scintillation vials and Dimilume (5 ml) added. Uptake of tritiated thymidine by cells was measured for 10 min on a Packard Tricarb liquid scintillation counter. The results were expressed as

J. B. Leiper & J. B. Solomon


c.p.m. minus background counts ± one s.e. and as the stimulation index (the ratio of mean experimental minus background counts/mean control minus background counts = E/C).



Plaque-forming assay Spleen cells from replicate wells cultured in vitro from 2-7 days, with or without sheep erythrocytes, were pooled and assayed for plaque-forming cells using essentially the method of Cunningham & Szenberg (1968).

. c -0 0




n7 2

RESULTS Number of macrophages in culture

The culture of guinea-pig spleen cells for 24 h prior to addition of PHA or SRBC greatly improved the reproducibility and responsiveness of the cells (Leiper & Solomon, in preparation). The presence of plastic-adherent (A) cells in cultures increased the mitogenic response of nonadherent (NA) cells two-fold. When the concentration of A cells was varied between the range 2 x 103 to 2 x 105 per culture the optimum response to PHA and SRBC occurred when 2 x 105 NA cells were cultured with 1-5 x 104 to 3 5 x 104 A cells (Fig. 1). Morphological examination of the added adherent spleen cells showed all cells were macrophages, no lymphocytes were observed. The larger number of macrophages may have partially inhibited the mitogenic response.

Figure 1. The effect of addition of aged adherent spleen cells on the mitogenic response (48 h) to PHA or SRBC of guineapig non-adherent spleen cells aged for 24 h; (0) 0-2 jug PHA; (v) I x 107 SRBC. Each point is the arithmetic mean from triplicate cultures.

Although PHA or SRBC induced only slight mitogenic responses in cultures of cells washed from the peritoneal cavity, A cells separated from peritoneal cavity washings were found to be quite as good as splenic A cells in supporting mitogenic responses when cultured with 2 x 1O5 NA spleen cells. The mitogenic requirements of splenic NA cells from foetal, juvenile and adult guinea-pigs for A cells in culture were compared. The ratio of NA to A

Table 1. Comparison of mitogenic responses of mixtures of non-adherent and adherent cells obtained from cultures, aged for 24 h of adult, juvenile and foetal guinea-pigs' spleen

Spleen cell type


Adult c.p.m.*

2 x 105 non-adherent


82+ 8 191+9 192+ 10 63+ 28 97+9 1 63+6 188+24 172+ 10 134+ 22 793+52 551+74

2x 104 adherent 2x 105 non-adherent with 2x 103 adherent

2x 105 non-adherent with 2x I04 adherent

Stimulation index

* c.p.m. (minus

2-3 2-3


Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs.

Immunology 1977 32 215 Ontogeny of mitogenic responsiveness to PHA and sheep erythrocytes and lymphokine production in foetal guinea-pigs J. B. LEIPE...
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