GM-CSF augments the immunosuppressive capacity of neonatal spleen cells in vitro

GM-CSF Augments the Immunosuppressive of Neonatal Spleen Cells in Vitro

Capacity

PHILIP J. MORRISSEY AND KOBIN IRELAND

Addition of exogenous granulocyte-macrophage colony stimulating lactor (GM-CSF) to cultures of adult murine spleen cells with sheep red blood cells (SRBC) results in an augmented plaque forming cell (PFC) response. The influence of GM-CSF on the ability of neonatal spleen cells to suppress the anti-SRBC plaque forming response of adult spleen cells was tested by adding GMCSF to cultures of neonatal and adult spleen cells. The suppressive capacity of the neonatal spleen cells was augmented by exogenous GM-CSF. The augmented suppression of the neonatal spleen cells was dependent on a G-IO adherent population since the addition of GM-CSF to cultures containing G-10 passed neonatal spleen cells resulted in an augmented PFC response and not suppression. Neonatal splenic glass adherent cells were also capable of suppressing the response. Neonatal spleen ceils or purified neonatal gIass adherent spleen cells cultured in the presence of GM-CSF had markedly increased levels of PGEz in the culture supernatant. Neonatat spleen cells cultured with GM-CSF had increased numbers ofmorphologically identifiable macrophages after 48 hr of culture. Both irradiation and G- 10 passageofthe neonatal spleen diminished the numbers of macrophages formed in response to GM-CSF. and both of these manipulations resulted in reversal of suppression in response to GM-CSF. Thus. the augmented suppressive capacity ot neonatal spleen cells in response to GM-CSF is probably mediated by its ability to drive monocyte to macrophage differentiation as well as increase the suppressive capacity of the existing neonatal splenic macrophages by increasing their production of PGE,. 1 ! YYI ZClldtmKPrev.I”<

IN-TRODIJC’HON

Spleen cells from newborn mace are immunologically inert and have been shown to be a potent source of cells which have the capacity to suppress the immune responses of normal adult spleen cells (1). Neonatal spleen cells have been shown to suppress the antibody forming response of adult spleen cells as well as their proliferative response to mitogens and alloantigens (2, 8). At least three different types of neonatal cells have been implicated in mediating this suppressive effect: T cells. non-T. non-B lymphocytes, and cells of the monocyte/macrophage lineage (2-g). Given the ease with which neonatal mice are made tolerant to non-self determinants, it has been postulated that the suppressive activity of these cells could be involved in the establishment of self tolerance (9). The cytokine GM-CSF’ stimulates the production of macrophages from hematopoietic precursors (10). Recently. it has been shown to have additional regulatory ’ Abbreviations used in this paper: GM-CSF. granulocpe-macrophage colony strmulatmg factor: P(;t,. prostaglandin E2: PFC. plaque forming cell: SAC, splenic adherent cell: SRBC. sheep red blood cell.

GM-CSF

AUGMENTED

IMMUNE

SUPPRESSION

463

influences on mature macrophages. For instance, GM-CSF has been shown to activate superoxide production in peritoneal macrophages, thereby increasing their microbicidal activity and induce tumoricidal activity in monocytes (11-13). Further studies have detailed the ability of GM-CSF to increase the PFC response of spleen cells to SRBC ( 14). It has been demonstrated that this enhancement of the PFC response is due to an increase in the antigen presenting capacity of the splenic macrophages. GM-CSF has also been implicated in the differentiation of functional antigen presenting cells from immature precursors (15, 16). Since GM-CSF is involved in the production and function of mature macrophages, we were interested in examining its effects on a population of less mature macrophages and macrophage precursors which are present in the neonatal spleen. It was possible that GM-CSF could either stimulate the maturation of these cells to functional antigen presenting cells, thereby diminishing their suppressive activity, or augment the suppressive function of these cells. The data presented here demonstrate that GM-CSF enhances the suppressive effect of neonatal macrophages and that the enhanced suppression correlates with an increase in the production of macrophages from monocyte precursors and the upregulation of the suppressive capacity of the existing neonatal macrophages via increased production of PGEz . MATERIALS

AND METHODS

,Wice. C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Timed pregnant C57BL/6J mice were purchased from The Jackson Laboratory or bred in our own colony from breeding pairs obtained from The Jackson Laboratory. Newborn mice were used at 3 to 5 days of age. Adult responder mice were female and between 12 and 16 weeks of age. Granulocyte-macrophage colony stimulating factor (GM-CSF). The gene encoding murine GM-CSF was cloned from an LBRM-33 cDNA library using the published cDNA sequence and was expressed in yeast (17, 18). GM-CSF was purified to homogeneity from the yeast culture supernatant by reversed-phase HPLC (19). The biological activity of GM-CSF was assessed by a murine bone marrow proliferation assay. The preparations routinely had an activity of 30-50,000 U/Fg protein where 1 U is defined as the amount of GM-CSF which causes 50% of the maximum [3H]TdR uptake ( 19). In vitro generation of anti-SRBCplaque-forming cells. Spleen cells ( 106) from normal adult C57BL/6J female mice were cultured with an equivalent number of SRBC and the indicated number of neonatal cells in 96-well flat-bottom plates (Costar, Cambridge, MA) in a volume of 0.2 ml. The culture media used was RPM1 1640 supplemented with 10% FCS, 5 X 10e5 M 2-ME, nonessential amino acids, sodium pyruvate, and L-glutamine. The cultures were incubated for 4 days at 37°C. Plaque forming cells (PFC) were enumerated using the slide modification of the Cunningham technique. Four culture wells per group were established and cultures wells were assayed individually. The data are presented as the geometric means : the standard error of the mean (SEM). The data presented are representative individual experiments which have been performed a minimum of three times with similar results. Depletion of adherent cells. Splenic adherent cells were depleted by passage over Sephadex G- 10 columns (Pharmacia, Piscataway, NJ) as previously described (20). Preparation of splenic adherent cells. Splenic adherent cells were isolated by adherence to glass as previously described (21). Briefly, 5 X lo7 cells were incubated on

glass petri dishes for one hour at 37°C‘. After washing, the plates were mcubated for an additional hour. After three additional washes, the adherent cells were removed by a brief incubation with EDTA (Versene 1:5000, GIBCO, Grand Island, NY). The ceils were then irradiated ( 1500R) and placed on a roller drum overnight at 37°C. The resulting population was between SOand 65% latex ingesting. Prustaglandin E2 measurement. PGE2 levels in 24-hr culture supernatants was measured using a competition radioimmunoassay (NEN Research Products. DuPont Co.. Wilmington, DE) according to the manufacturer’s recommended procedure. The rabbit anti-PGE? antibody used in this assay exhibited minimal cross reactivity (~3%) with other members of the prostaglandin family (e.g., PGE, . PGA?, etc.). PGE2 concentrations in the culture supernatants were estimated by comparison with a standard curve generated with known PGEz standards.

Response h.v Neonatal Spleen C’eIh Previously, we have demonstrated that exogenous GM-CSF augmented the primary in vitro anti-SRBC response by adult spleen cells and that this effect was mediated by an enhanced antigen presenting function of the splenic adherent cells (14). We were similarly interested in the effects of exogenous GM-CSF on the suppression of this response by neonatal spleen cells. It can be seen in Table 1A that the addition of spleen cells from 4-day-old mice to adult spleen cells suppressed the anti-SRBC PFC response in a dose dependent manner. In this experiment, the adult spleen cells cultured with SRBC generated 147 PFC and this response was decreased to 62 PFC by the addition of 4 X lo5 neonatal spleen cells at the initiation of culture. This suppression

Suppression

Responding A.

spleen

of the Primary

1.4 X 10b adult 1O6 adult

1).

1.4 X 10’ adult 10” adult (+lL-.!j --l__--

Response

by Neonatal

Number 0t’ neonatal spleei~ cells added

cells

I O6 adult

B. C.

I’FC

Spleen

GM-CSF

Ceils Is Augmented

PI-i‘ culture“

by GM-C%

Percentage suppression’

?iOW

_._

10’ .2 .A 10’ 4 x IO lione None !Oi z , I(!< ‘1 ‘.’ (0 None 4 A’ IO’ -__--“.--..-_-

..---

4 ^... - _--__--

’ The data are the geometric means of four indtvidually assayed culture wells (YSEM). Neonatal spleen cells cultured withy SRBC ( 106) with or without GM-CSF generated no PFC. b Percentage suppression is calculated as 1~~ (response of adult spleen with neonatai celis/response of adult spleen cells alone) X 100. The concentration of GM-CSF and IL-2 in the cultures was 15 rig/ml.

GM-CSF AUGMENTED

IMMUNE

465

SUPPRESSION

TABLE 2 Augmented Suppression by GM-CSF Is Dependent on G- 10 Adherent Cells Neonatal spleen cells added (X 1O-‘y A. None 2 4 B. None 2 4 C. 1 (G-10 passed) 2 4 D. 1 (G-10 passed) 2 4

GM-CSF

PFC/culture

-

112 (1.04) 62 (1.07) 32 (1.12) 12 (2.60) 187 (1.16) 56 (1.08) 33 (1.17) 10 (2.04) 52 (1.04) 27 (1.13) 26 (1.60) 165 (1.07) 206 (1.13) 235 (1.19)

+ + + + + + +

Percentage suppression

45 71 89 70 82 91 53 76 77 0 0 0

iI Adult spleen cells ( 106) were cultured with SRBC ( 106) and the indicated number of untreated or G- 10 passed neonatal spleen cells with or without 15 rig/ml of GM/CSF. The data are the geometric means (FSEM) of four culture wells. The response of 1.4 X lo6 adult spleen cells (density control) averaged 125 PFC/culture.

was not due to an increased cellular density of the cultures since the response of 1.4 X 1O6 adult spleen cells, which was the number of cells contained in the adult ( lo6 cells) plus neonatal spleen (0.4 X lo6 cells) cultures, was 128 PFC (Table 1B). It is also shown in Table 1C that the addition of GM-CSF to cultures of adult spleen cells and SRBC augmented the PFC response by almost twofold. The addition of neonatal spleen cells to these cultures (containing GM-CSF) suppressed the response in a dose dependent manner. Indeed, the inhibition of the response was greater in the presence of GM-CSF (88%) than in the absence of GM-CSF (58%) despite the almost twofold augmentation of the response of the adult spleen cells alone by GM-CSF. Addition of exogenous recombinant IL-2 to the cultures containing 4 X lo5 neonatal spleen cells plus GM-CSF resulted in a dramatic increase in anti-SRBC PFC response (Table 1D). Thus, these results demonstrate that exogenous GM-CSF, although augmenting the primary anti-SRBC response of normal adult spleen cells, was not able to overcome the suppressive influence of the added neonatal spleen cells. In fact, the results indicate that GM-CSF augments the suppressive capacity of the neonatal spleen cells. Addition of IL-2 overcomes the augmented suppression mediated by GM-CSF. The Augmented Suppression of Neonatal Spleen Cells by GM-CSF Is Dependent on a G- 10 Adherent Cell It is well appreciated that neonatal spleen cells contain at least two types of suppressor cells; one of lymphocytic origin and the other of the monocyte/macrophage lineage (4-7). In order to ascertain which population was responsible for the augmented suppression mediated by GM-CSF, neonatal spleen cells were passed over a Sephadex

MOKRISSl2

466

AND

IRELANI

!

G- 10 column which would retain adherent cells, and then their suppressive capabilities alone and in the presence of GM-CSF were assessed. The results of a representative experiment are shown in Table 2. Here it can be seen that the PFC response of the adult spleen cells was suppressed by the addition of whole neonatal spleen cells (Table 2A). The addition of GM-CSF (Table 2B) to the cultures of adult cells alone increased the response from I I2 to 187 PFC/culture and as seen previously, the addition of neonatal spleen cells to the adult cells in the presence of GM-CSF suppressed the response. Of interest was the fmding that the G-10 passed neonatal spleen cells were still able to suppress the PFC response (Table 20 However. in contrast to the effects of GM-CSF on the suppressive capacity of unfractionated neonatal spleen cells, GMCSF augmented the PFC response when G- 10 passed neonatal spleen cells were added to the cultures (Table 2D). Thus, the G-LO passed neonatal spleen cells were still able to suppress the PFC response of the adult spleen but the addition of exogenous GMCSF overcame the suppressive influences of the G- 10 passed neonatal spleen cells.

It is w-e11appreciated that G-10 Sephadex columns largely retain an adherent macrophage/monocyte cell population (20). In order to determine if this population was indeed suppressive, we prepared adherent cells from neonatal spleens and assessed their suppressive capacity on the anti-SRBC PFC response of adult spleen cells. Adherent cells were prepared by incubating neonatal spleen cells on glass petri dishes for 2 hr at 37°C. The plates were washed extensively and the remaining adherent cells removed by a brief incubation with EDTA. The cells were then irradiated (I 500 R) and placed on a roller drum at 37°C overnight. The next day the cells were washed, counted, and added to the cultures of fresh adult spleen cells. The results of a typical experiment are shown in Table 3. Here it can be seen that the unfractionated neonatal spleen cells were capable of suppressing the PFC response of the adult spleen cells (Table 3A). When the neonatal splenic adherent cells (SAC) were added to the cultures. the response was suppressed to a greater degree (Table 3B). For instance, addition of 2 X IO5 neonatal spleen cells suppressed the response by 59% and the addition of 2

Adherent __~_-.-.-.~.-.--.--_ Cells added A. None Neonatal Neonatal Neonatal B. Neonatal Neonatal C. GM-CSF

Cells t?om Neonatal Spleen Cells IMediate SuppressIon Which Is Augmented by Pulsing with GM-CSF ._---_______. ____

to IO6 adult

spleen

ceik

Percen taye ruppression

spleen ( I X 10’) spleen (2 Y li15J spleen (4 x IO’/ SAC ( I X 105J SAC 13 ,\Y ii)‘1 pulsed neonatal SAC‘ (I .q 1:J‘)

” Adult spleen cells were cultured with 10’ SRBC plus the indicated additions. The data are the geometric means of four individually assayed cultures (XSEM). The response of 1.4 X lo6 adult spleen cells to SRBC was 396 PFC/culture (density control).

GM-CSF AUGMENTED

IMMUNE

467

SUPPRESSION

X lo5 SAC suppressed the response by 84%. Thus, the neonatal splenic adherent cells were generally more efficient in suppressing the response than whole neonatal spleen cells. It should be noted that the addition of 2 X lo5 SAC from adult mice to the cultures had a minimal augmenting effect (data not shown). Thus, the suppression was not due generally to an excess of macrophages in culture. We have previously shown that pulsing adult SAC with GM-CSF for 10 min at 37 “C and then washing extensively resulted in an increased antigen presenting capacity when they were added to adherent cell depleted adult spleen cells (14). We performed a similar experiment with neonatal SAC. These cells were pulsed for 10 min with GMCSF (100 ng), washed three times, counted, and added to adult spleen cell cultures. In Table 3C it can be seen that the GM-CSF pulsed adherent cells had a greater suppressive capacity than the same number of unpulsed neonatal splenic adherent cells. These results demonstrate that the adherent cell fraction of neonatal spleen cells could suppress the PFC response of adult spleen cells and pulsing them with GMCSF prior to culture augmented their suppressive capacity. Thus, it appears that GMCSF can increase the suppressive capacity of the adherent neonatal spleen cell. GM-CSF Induces Increased Levels of PGE, in Cultures of Neonatal Spleen Cells Macrophage produced PGE2 has well known immunosuppressive effects on in vitro responses (22). Thus, it was of interest to determine if GM-CSF could induce augmented PGE2 production by neonatal spleen cells. Neonatal spleen cells ( 106) were cultured for 48 hr with or without GM-CSF. PGE2 levels were measured in the culture supernatants by RIA. Data detailed in Table 4 show that the level of PGE2 in the supernatant of cultured neonatal spleen cells was more than doubled by the addition of GM-CSF. When neonatal spleen cells were cultured with antigen (SRBC) the constitutive level of PGE2 was increased and the addition of GM-CSF to the cultures resulted in more than a fourfold increase in PGE2 levels. In order to determine more precisely if the increased PGE2 levels in response to GM-CSF could be attributed to the adherent population of neonatal spleen cells, glass adherent neonatal cells were isolated and cultured with or without GM-CSF. In these cultures the level of PGE2 was increased in response to GM-CSF. In comparison, the addition of GM-CSF to cultures of adult TABLE 4 Production of PGE2 by Neonatal Spleen Cells and Splenic Adherent Cells Is Increased by GM-CSF

PC& (PO-4 Culture”

Without GM-CSF

With GM-CSF

Neonatal spleen cells Neonatal spleen cells + SRBC Neonatal SAC Adult SAC G- 10 passed neonatal spleen

300 550 550 250 200

600 2500 1830 250 250

a G- 10 passed or whole neonatal spleen cells ( 106) or splenic adherent cells ( 105) were cultured for 48 hr in 24-well Costar plates in 1 ml. Culture supematant was removed and assessedfor PGEz levels by RIA. The results are the means of culture wells assayed individually.

splenic adherent cells did not result in increased PGE? levels. Adherent cell depleted neonatal spleen cells (G-10 passed) showed a minimal increase in PGE? levels in response to GM-CSF.

The neonatal murine spleen is primarily a hematopoietic organ (23). It IS possible that GM-CSF could augment the suppressive effect of neonatal spleen cells by driving the production of macrophages from immature precursors and thus increasing the number of macrophages in the culture (5). This hypothesis was tested by determining the effect of irradiation on their suppressive capacity and responsiveness to GM-CSF. The results of a representative experiment are shown in Table 5. Here it can be seen that neonatal spleen cells suppressed the anti-SRBC response of the adult spleen cells (Table 5A) and that this suppression was augmented by the addition of GM-CSF (Table 5B). The addition of irradiated neonatal spleen cells was also suppressive (Table SC) although less so than unirradiated cells. Addition of GM-CSF to cultures with irradiated neonatal spleen cells (Table SD) resulted in an augmentation of the PFC response. In parallel cultures. neonatal spleen cells stimulated with GM-CSF manifested a strong proliferative response (in a typical experiment, 63,875 cpm of [‘H]TdR incorporation with a background (cells cultured in the absence of GM-CSF) of less than 1000 cpm). Irradiated (2000 R) neonatal spleen cells did not proliferate in response to GM-CSF significantly over the background response (< 1000 cpm). Thus. the decreased proliferative response correlated with the ability of GM-CSF to overcome the suppressive effect of the irradiated neonatal spleen cells.

Augmented

Suppression

Neonatal spleen cells added”

bq Neonatal

Spleen

Cells in Response

to GM-CSF

is Radiation

Sensitive

Percentage suppression

” .4dult spleen cells were cultured with SRBC and the indicated number of neonatal spleen cells 111 the presence or absence of 15 rig/ml of GM-CSF. The data are the geometric means (* SEM) of four individually assayed culture wells.

GM-CSF AUGMENTED

IMMUNE

469

SUPPRESSION

G-,10 Passage or Irradiation of Neonatal Spleen Cells Decreases the Production of Macrophages in Response to GM-CSF The effects of G- 10 passage and irradiation on the number of macrophages formed from neonatal spleen in response to GM-CSF was also investigated (Table 6). Unmanipulated, G- 10 passed, or irradiated neonatal spleen cells were cultured in 24-well plates (2 X lo6 cells in 1 ml) for 48 hr. At this time the wells were washed extensively with PBS to remove nonadherent cells and then stained with Wright’s Giemsa stain. The number of morphologically identifiable macrophages was enumerated microscopically and the average number of cells/field is reported. These results (Table 6) indicate that both G-10 passage and irradiation significantly decreased the numbers of morphologically identifiable macrophages formed in these cultures in response to GM-CSF. DISCUSSION The suppressive capacity of murine neonatal spleen cells has been well documented, but the exact lineage of the suppressor cells and the nature of their regulation has not been decisively determined (l-8). In this report we have provided data which differentiate components of the suppressive mechanism by the ability of exogenously added GM-CSF to enhance the suppression or overcome it. We have previously shown that GM-CSF when added to adult spleen cell cultures with SRBC caused an enhanced PFC response by augmenting the antigen presenting capacity of the macrophages ( 14). Thus, we were interested in determining effects of GM-CSF on the suppressive capacity of neonatal spleen cells. It was possible that GM-CSF might induce the differentiation or maturation of the suppressive macrophage to a nonsuppressive, functional antigen presenting cell. However, our data suggest that this was not the case, since exogenous GM-CSF augmented the suppressive capacity of the neonatal spleen cells. The initial indication that the action of GM-CSF in this system was on cells of the monocyte/ macrophage lineage was revealed by the effect of passing the neonatal spleen cells through a Sephadex G-10 column. G-10 passed neonatal cells were still suppressive, but in contrast to whole neonatal spleen cells the addition of GM-CSF to the cultures did not enhance the suppression but overcame it, resulting in an augmented response. TABLE 6 -

Effect of G- 10 Passage or Irradiation on the Differentiation of Macrophages from Neonatal Spleen No. of macrophages/field Cells cultured with

-

Cells”

GM-CSF

No GM-CSF

Neonatal spleen G- 10 passed neonatal spleen Irradiated (2000 R) neonatal spleen

49.5 (4.8) 21.7 (2.9) 14.8 (2.75)

23.3 (2.4) 14.6 (1.8) 10 (1.35)

’ Whole, G-10 passed, or irradiated neonatal spleen cells were cultured for 48 hr in 24-well plates (2 X lo6 cells/well). The wells were washed and stained. The number of morphologically identifiable macrophages were enumerated microscopically. The data are the arithmetic mean (-tSEM) of at least 15 fields (magnification, 40X).

Since it is well known that cells of the monocyte/macrophage lineage are adherent to G- 10, this result suggested that the enhanced suppression seen by the addition of GMCSF to cultures containing neonatal spleen cells was due to its effects on monocytesj macrophages. In order to characterize the G-10 adherent cell which was responsible for the enhanced suppressive response to GM-CSF, we first isolated glass adherent neonatal spleen cells. When these cells were added to cultures of adult spleen cells they were suppressive and their suppressive nature was enhanced by pulsing the cells with GMCSF. This indicated that macrophages from neonatal spleens were indeed suppressive in and of themselves and that GM-CSF did not cause a maturation into antigen presenting cells but enhanced their suppressive capacity. The mechanism of this macrophage mediated suppression may be through the elaboration of high levels of PGE. It has been previously observed that PGE? could decrease the primary response to SRBC (24). Measurement of PGE, levels in cultures of neonatal spleen cells revealed that addition of GM-CSF results in increased PGE? levels. Indeed, the greatest levels of PGE? were seen in response to GM-CSF plus antigen (SRBC). Purified neonatal SAC cells showed increased PGE:! levels in response to GM-CSF but neither adult SAC nor G-10 passed neonatal spleen cells did. These results are consistent with the hypothesis that the mechanism of suppression mediated by the neonatal macrophage is via increased PGEz production. Indeed. PGEi is well known for its immunosuppressive properties, especially for its ability to inhibit IL.-Z production (25-27). Our results demonstrating that exogenous IL-2 can overcome the suppressive activity of the neonatal macrophage are also consistent with a PGE: mediated mechanism (25). Our results also suggest that the influence of GM-CSF on the suppressive capacities of neonatal spleen cells involves the stimulation of proliferation and the differentiation of macrophages from precursor cells. Abrogation of the proliferative response of the neonatal spleen cells by irradiation resulted in the reversal of suppression by the addition of exogenous GM-CSF. It should be noted that irradiated neonatal SAC were still capable of secreting PGE: since these cells were irradiated prior to culture for PGE, measurement (Table 4). These results indicated that the GM-CSF driven proliferation and differentiation might have contributed significantly to the suppressive tendencies of neonatal spleen cells and that this function of GMCSF was involved in the enhanced suppressive capabilities of neonatal spleen cells. Indeed the observation that irradiation decreased the formation of identifiable mace, rophages in response to GM-CSF in i’itm also supports this contention. Thus. the augmentation of suppression in this system by GM-CSF is a complex event depending on the presence of neonatal adherent cells and nonadherent cells which respond to GM-CSF to form macrophages. Elimination of the adherent cells by G- 10 passage or the abrogation of the proliferative response by irradiation sufficiently compromised the suppressive abilities and allowed the enhancing effect of GM-CSF on antigen presenting cells to prevail. In support of our findings. previous studies have shown that the suppressive influence of neonatal spleen cells was due to the differ.entiation of macrophages from monocytic precursors. thereby increasing the total number of macrophages present in the cultures (5). Our data extend those results and demonstrate that GM-CSF may be a primary regulatory molecule for this differentiative step. GM-CSF has been shown to be a potent regulator ot’ macrophage functton. ‘1hus. GM-CSF has been shown to cause increased Fc receptor expression, increased B.-i

GM-CSF AUGMENTED

IMMUNE

SUPPRESSION

471

production, induction of tumoricidal and microbicidal activity, and increased PGE2 synthesis in LPS primed monocytes (1 I- 13, 28, 29). Also it has been shown that in precultured adult bone marrow, GM-CSF induces the expression of la antigens and antigen presenting function ( 15, 16). The data presented here suggest that GM-CSF is not capable of causing the differentiation of existing macrophages in the neonatal spleen or their monocytic precursor into functional antigen presenting cells. It may be that the ability of macrophages to develop into functional antigen presenting cells occurs later in ontogeny. Indeed, it has been reported that the expression of la antigens by macrophages in neonatal mice is down-regulated by a splenic suppressor cell (30). ACKNOWLEDGMENTS ‘The authors gratefully acknowledge the expert technical assistance of Linda Bressler, and thank Linda Troup and Debbie Wright for the expert preparation of the manuscript.

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