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Ontogeny of macrophage function I. Phagocytic activity and A-cell activity of newborn and adult mouse peritoneal macrophages
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Printed in the United States
ONTOGENY I.
PHAGOCYTIC
NEWBORN
KOJI
NAKANO~,
AND
Vol. 2, pp. 505-518, 1978 Pergamon Press, Inc.
OF MACROPHAGE ACTIVITY
ADULT
TOMOHIDE
MOUSE
AND
FUNCTION A-CELL
ACTIVITY
PERITONEAL
HOSOKAWA§
AND
OF
MACROPHAGES*
SHIGERU
MURAMATSU¶
Department of Zoology, Faculty of Science, Kyoto University, Kitashirakawa-0iwakecho, Sakyo-ku, Kyoto, Japan 606
ABSTRACT. Phagocytic activity and immune-participating A-cell acitivity of newborn and adult mouse macrophages were investigated under in vi2AO conditions. Thioglycollate medium-stimulated newborn (SNB) or adult (SA), and nonstimulated adult (NA) peritoneal macrophages were used. Immune complexes of sheep erythrocytes (SRBC), aldehyde-fixed SRBC, and latex beads were employed in phagocytosis tests. A-cell activity was estimated as the capacity to support primary or secondary responses of macrophage-deprived adult spleen cells to SRBC. Results obtained were: i) phagocytic activity of NA macrophages was highest and that of SNB macrophages was higher than SA macrophages, 2) no difference was observed in A-cell acitivity for secondary response among SNB, SA, and NA macrophages, and 3) SNB macrophages were lacking in A-cell activity for primary response. These results suggest that the substantial A-cell function should be evaluated in primary responses, and that the A-cell and phagocytic functions do not necessarily accompany each other.
INTRODUCTION Newborns of mice and other animals are known to be immunologically immature (i). In adult animals, thymus-derived T lymphocytes, nonthymus-derived B lym-
* Supported by grant in aid for scientific research from the Ministry of Education, Science and Culture of Japan. Visiting investigator from the Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto. § Present address: Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kyoto. ¶ To whom correspondence should be sent. 01451305X17810701-05051502.0010 Copyright© 1978 Pergamon Press
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phocytes, and macrophages as accessory cells (A-cells) are involved in the immune responses (2,3). Interaction among T lymphocytes,B lymphocytes and A-cells are necessary for antibody responses to so-called thymus-dependent antigens (2-4). Thus, immunological unresponsiveness of newborn animals must be attributable to the immaturity of at least one or at most all of these three categories of cells. According to some authors, the ontogenetic differentiation of B cells may precede that of T cells (5-9). On the other hand, inoculation of adult peritoneal exudate cells (PEC) into newborn mice (i0,ii), rats (12) or rabbits (13) slightly but significantly enhanced the antibody response. In another experiment (14), only adult PEC, but not newborn PEC, were effective in restoring immune responsiveness of sublethally irradiated adult mice. These reports, however, do not seem to give evidence that the deficiency of macrophages in A-cell function is mainly responsible for immunological unresponsiveness of newborn animals, since lymphocytes may be contained in adult PEC preparations. Phagocytosis* of foreign materials is, undoubledly, the most fundamental and basic function of macrophages, a universal characteristic of the entire animal kingdom (15). By comparison, lymphocyte-stimulating A-cell activity seems to be relatively refined and more phylogenetically evolved function. Therefore, it may be problematical whether A-cell activity is directly related to phagocytic acitvity, or whether these two activities are intrinsically independent of each other. In this study, we investigated phagocytic activity and A-cell activity of peritoneal macrophages of newborn and adult mice. METHODS Mice : C3H/HeMs mice of both sexes (supplied from the Institute of Laboratory Animals, Faculty of Medicine, Kyoto University) were used. The age of adult mice in these experiments was about 2 months. AKR mice were used as a source of C5 -deficient serum and as anti-Thy 1.2 antibody producers. Peritoneal exudate cells (PEC) : Normal adult (NA), thioglycollate (TGC)-stimulated newborn (SNB), and adult (SA) mice were killed by exsanguination from the carotid artery and their peritoneal cavities flushed aseptically with Eagle's minimum essential medium (MEM) using plastic syringes. PEC were spun down at i~000 rpm for 5 minutes and resuspended in MEM with 10% fetal bovine serum (FBS, Flow Laboratories). TGC (Brewer's medium, Difco Laboratories) was injected intraperitoneally in the volume of 2 ml into adults or 0.2 ml into neonates (within one day after birth) 4 days before harvest of PEC. Preparation of macrophages for phagocytosis test : Glass petri dishes (60 mm diameter) including 8 rectangular glass cover slips (22 x i0 mm) on their bottoms were added with i x 106 PEC in the final volume of 5 ml, and kept for 22-24 hours at 370C in a C02 incubator (5% C0295% air). After then cover slips were washed thoroughly with MEM to remove nonadherent cells and FBS. Two cover slips each were transferred to 35 mm glass petri dishes containing 2 ml of MEM without FBS.
*
The term "phagocytosis" is used to include not only ingestion of test particles by macrophages, but also their attachment onto macrophage receptors.
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Erythrocytes : Sheep erythrocytes (E or SRBC) in Alsever's solution (Nikken Animal Blood Center, Kyoto) were washed three times with saline (0.15M NaCI) and suspended in MEM at 108 E/ml as the test particle preparation for phagocytosis test. Glutaraldehyde-fixed E (FE) and latex beads : Washed E were fixed with 1% glutaraldehyde, repeatedly washed and stored at 4°C in saline at 109 cells/ml until use. As test particles, FE were suspended in MEM at 108 cells/ml. Polystyrene latex beads (i.011 ~m diameter, Dow Chemical Co.) were suspended at a concentration of 0.1%. Antisera : Rabbit anti-E serum was obtained one week after intravenous injection of 2 x 109 E. IgG and IgM fractions of the antiserum were prepared by means of a Sephadex G-200 column. Anti-Thy 1.2 serum was prepared by immunizing AKR mice with C3H mouse thymocytes (16). Preparation of E-IgG complex (EA) : EA and EAC (see below) were prepared essentially according to the method of Bianco et al. (17). The i:i mixture of E suspension (109/ml) and anti-E IgG at a final concentration just below aggulutinating E was incubated with gentle shaking at 37°C for 15 minutes. After washing three times with MEM, EA thus prepared was suspended at 108 cells/ml. Preparation of E-IgM-C3 (EAC) and ghost EAC (GEAC) : E-IgM complex was prepared similarly to EA, and suspended at 109/ml in veronal-buffered saline containing glucose, Ca+2, Mg+2 and gelatin. Mixture at i:i of the E-IgM and i/i0 diluted fresh AKR serum was incubated at 37°C for i0 minutes. EAC thus prepared was washed three times with MEM and suspended at 108 cells/ml. E-IgM and AKR serum-treated E were also prepared as control test particles. Neither attachment nor ingestion of these particles was observed. GEAC was used for blocking C3 receptors on macrophages. Washed E were lysed in hypotonic phosphate buffer solution (18) and hemoglobin-free ghosts were obtained by repeated washing with the buffer by centrifugation at 15,000 rpm for 30 minutes. Ghosts were homogenized by a supersonicator, sedimented by centrifugation at 30,000 rpm, and resuspended in MEM at the concentration equivalent to 2 x 10 I° E/ml. Then, GEAC was prepared following the procedure similar to that for EAC, washed three times with MEM by centrifugation, and diluted to the concentration equivalent to 2 x 109 E/ml. Aggregated human gamma globulin emulsion (aHGG) : Human gamma globulin (Fraction II, Wako Purechemical Industry, Osaka) at a concentration of 50 mg/ml in saline was heated at 63°C for 20 minutes and centrifuged at 3,000 rpm for 30 minutes. Unsedimented fraction of aHGG was used for blocking Fc receptors on macrophages. Assay system for phagocytic activity : To each 35 mm glass petri dish containing two cover slips with macrophages and 2 ml of MEM without FBS was added 0.2 ml of one of the test particle preparations, and incubated for 120 minutes (see Fig. 2). Usually the dishes were incubated at 4°C for the attachment of particles onto macrophage receptors without ingestion, and at 37°C for the ingestion of particles. After incubation, cover slips were washed several times with shaking and fixed with 2% gutaraldehyde in MEM. After washing in distilled water, cover slips were reversed upside down, and embedded on slides with glycerin jelly. Macrophages were observed by negative phase contrast microscopy. To examine the ingestion
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of EA and EAC by macrophages, cover slips were dipped into 0.83% NH~CI for 5 minutes to remove extracellularly attached erythrocytes before fixation. In each test, at least 400 macrophages were exmined. The data concerning phagocytosis are presented in this paper according to Bianco et al. (17). Percent ingestion or attachment is the percentage of macrophages which ingested or attached test particles. Ingestion or attachment index is the number of particles ingested ar attached per i00 macrophageso The attachment was regarded as positive when one macrophages attached three or more EA or EAC. Experiments were repeated at lest three times for each preparation of test particles, and representative results are shown in this paper. Blockade of Fc receptors by aHGG and C3 receptors by GEAC : Cover slips with macrophages in 35 mm petri dishes were preincubated in 2 ml of MEM containing lO mg of aHGG or 0.6 ml of GEAC at 37°C for 30 minutes, and then test particles were added without changing the medium. Trypsin treatment of macrophages : To destroy selectively C3 receptors (17), cover slips with macrophages were incubated at 37°C for 20 minutes with 1 mg/ml of trypsin (Sigma Chemical Co.) in MEM. Cover slips were washed twice with i mg/ml of soybean trypsin inhibitor (Worthington Biochemical Co.) in MEM to terminate trypsin digestion, and submitted to the phagocytosis test in fresh MEM. Test for the A-cell activity in anti-SRBC response in vitro : A-cell activity of macrophages was assessed by the capacity to restore the in vig~o anti-SRBC response of macrophage-deprived spleen cells as described below. Culture medium was MEM with 2 mM L-glutamine and 10% FBS. In the case of dish culture, 5 x 10-SM 2-mercaptoethanol was added to the medium (20). Viability of tells was determined by the trypan blue dye exclusion test. PREPARATION OF LYMPHOCYTE-DEPLETED MACROPHAGES. We depleted lymphocytes from PEC by X-irradiation and anti-Thy 1.2 treatment without detriolating A-cell activity of macrophages (3,22). Normal and TGC-stimulated mice received whole body X-irradiation at 1,300 R with a therapeutic X-ray emitter (Toshiba Electric CoL, Tokyo). Irradiation was performed at 180 kV and 20 mA, target distance 44 cm, with 0.5 mm Cu and 0.5 mm AI filter, at the rate of i00 R/min. After irradiation, peritoneal cells were collected and treated with anti-Thy 1.2 serum and complement (guinea pig serum absorbed on C3H mouse spleen cells) (21), washed, and resuspended in the culture medium. PREPARATION OF MACROPHAGE-DEPRIVED SPLEEN CELLS. Normal or primed spleen cells were passed through Sephadex G-10 (Pharmacia Fine Chemicals) column according to the method of Ly and Mishell (23). Primed spleen cells were obtained from mice injected intravenously with 107 SRBC 4 days previously.
IN VITRO ANTI-SRBC RESPONSE. Marbrook's flasks (24) (Takahashi Giken, Tokyo) and plastic dishes (Falcon 3001, diameter 35 mm, Falcon, Oxnard) were employed. In primary responses, only Marbrook's flaskswere used. The test system usually contained 107 macrophage-deprived spleen cells, 5 x 106 SRBC, and 104-106 macrophages. Volumes of the culture medium were i ml in the inner chamber and i0 ml in the outer chamber of Marbrook's flasks, and 3 ml in plastic dishes. Cultures were maintained at 37°C in 5% C02 and 95% air without rocking for 4 days to obtain peak responses. Antibody-forming cells were assessed by the plaque-forming cell assay (25) in agarose gel on slides (26). Only direct plaque-forming cells (PFC) were detected in these experiments. Results are presented as the mean number of duplicate cultures.
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RESULTS Determination of optimum timing for collecting peritoneal macrophages for the phagocytosis test after TGC injection : PEC were collected on various days after injection of TGC into adult mice, cultured for 24 hours, and submitted to phagocytosis test using EA as test particles. As shown in Fig. i, both attachment and ingestion indices were highest in normal macrophages (day 0) and they fell off rapidly i day after TGC injection. The indices tended to increase again up to the gentle peak on day 3 to day 5. Thus, we decided to collect PEC 4 days after TGC injection as the source of SA and also SNB macrophages.
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FIG. I Phagocytic activity of adult peritoneal macrophages collected on various days after TGC injection. Macrophages cellected on day 0 were TGC-unstimulated cells. Open and closed circles indicate, respectively, attachment and ingestion indices for EA. Figures in parentheses are percentages of macrophages carrying EA.
Kinetics of phagocytosis after addition of test particles : SA and NA macrophages were given EA and EAC at 4°C or at 37°C. At various times thereafter, attachment and ingestion of test particles were evaluated. As depicted in Fig. 2, both attachment and ingestion indices tended to increase until 2 hours following the addition of test particles. Thus, the phagocytosis test was performed for 2 hours in the following experiments. Phagocytic activities : Fig. 3 shows the ingestion of E, FE, and latex beads by NA, SA, and SNB macrophages. FE and latex were actively ingested at 37°C but the phagocy~osis of E was practically nil in any macrophage preparation. Ingestion indices for FE and latex were highest in NA, second in SNB, and lowest in SA macrophages. Attachment of these three kinds of particles on macrophages did not occur either at 37°C or at 40C. Results of experiments to investigate the phagocytic activity for EA and EAC are shown in Fig. 4. As in the case of FE and latex, NA macrophages were more phagocytic than the other two macrophage preparations, especially in the phagocytosis of EAC. In the data shown in Fig. 4, attachment and ingestion indices of NA macrophages were more than eight times and two times, respectively, as much as those of SA macrophages. Percentages of NA macrophages attaching or ingesting EAC were also remarkably higher than
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those of SA macrophages. Difference between SNB and SA macrophages was not so conspicuous, but any values of indices of SA macrophages did not surpass those of SNB macrophages.
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Time course of the phagocytic activity after initiation of SA or NA culture with EA or EAC. Open and closed symbols indicate, respectively, attachment (upper panel) and ingestion (lower panel) indices for EA or EAC. Circles and squares represent, respectively, indices of SA and NA macrophages. Figures in parentheses are percentages of macrophages carrying EA or EAC.
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INCUBATIONTIME (MIN)
Effect of aHGG~ GEAC and trypsin on attachment and ingestion EA and EAC : To confirm that EA was phagocytosed via Fc receptors on macrophages, blocking of receptors with aHGG was attempted before the addition of EA. Similarly, preincubation of macrophages with GEAC and pretreatment with trypsin were performed to block or digest C3 receptors. As shown in Fig. 5, both attachment and ingestion of EA were inhibited only by aHGG but not by GEAC or trypsin, and those of EAC were inhibited by GEAC or trypsin but not by aHGG. These results seem to indicate that EA and EAC were phagocytosed via different receptors, probably Fc receptors for EA and C3 receptors for EAC. Ingestion of latex particles was not affected by GEAC nor trypsin, but the ingestion index was somewhat reduced by aHGG. This does not seem, however, to be caused by masking of Fc receptors with aHGG, since percentage of macrophages ingesting latex beads was not reduced, as contrasted with the remarkable reduction of EA ingestion not only in the ingestion index but also in the percentage. A-cell activity in A-cell activity capacity to support cells. Experiments
secondary response : of macrophages in secondary response was estimated by their in v a ~ O antibody response of SRBC-primed spleen lymphoid were repeated five times and similar results were obtained,
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C3H 1400
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1200
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600 83
z
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FIG. 3 Ingestion indices of SNB, SA and NA macrophages for E, FE, and latex beads. Height of each column refers to the value in index. Figures on columns are percentages of macrophages ingesting test particles.
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FIG. 4 Attachment (open column) and ingestion (shaded column) indices for EA (left panel) and EAC (right panel) of SNB, SA and NA macrophages. Figures on columns are percentages of macrophages carrying test particles.
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[]
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CONTROL
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Effects of the pretreatment of macrophages with aHGG, trypsin, or GEAC on the attachment at 4°C or at 37°C (upper panel) and ingestion at 37°C (lower panel) of EA, EAC, or latex beads. NA macrophages were used in this experiment. Procedures in detail are in the text. Height of each column represents attachment or ingestion index, and figures on columns are percentages of macrophages carrying test particles.
so that the representative result is shown in Fig. 6. It is indicated that deprivation of macrophages from spleen cell preparation resulted in reduction in the number of PFC when compared with original cell preparations; any one preparation of lymphocyte-depleted macrophages effectively supported the antibody response of such primed lymphocytes. In dish cultures, the number of added macrophages was changed from i0 ~ to 106 , but no dose effect was observed. In a preliminary experiment, when only macrophages were cultured with antigen, no PFC were detected (data not shown). A~cell activity in primary response : Unprimed spleen cells deprived of macrophages were used to estimate A-cell activity of macrophages in primary in vi~YLo responses. In the experiment of Fig. 7, unirradiated and anti-Thy 1.2-untreated PFC were used. Lymphocytedepleted macrophages were used in the experiments of Fig. 8. In both Fig. 7 and Fig. 8, adult 105 and 106 macrophages (SA and NA) were able to reconstitute partly or completely i0 ? macrophage-deprived spleen cells effecting responses to SRBC. In experiment 3 (Fig. 8), an excess of SA macrophages (106 ) was ineefective in supporting the response. It should be noticed that A-cell activity of newborn macrophages (SNB) was very low in comparison with adult macrophages. This was the case both with lymphocyte-depleted macrophages (Fig. 8) and with the original PEC (Fig. 7).
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2 ° RESPONSE (£xP.1)
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In V~;C~LO s e c o n d a r y a n t i - S R B C r e s p o n s e of m a c r o p h a g e - d e p r i v e d s p l e e n cells suppl e m e n t e d w i t h SA, NA, or SNB m a c r o p h a g e s . H e i g h t of each c o l u m n refers to the n u m b e r of PFC per c u l t u r e (the g e o m e t r i c a l m e a n of d u p l i c a t e cultures) in p l a s t i c dish (left panel) or M a r b r o o k ' s f l a s k (right panel). O p e n c i r c l e s rep r e s e n t the a r i t h m e t r i c a l m e a n of d u p l i c a t e assays for each culture. Sources and n u m b e r s of cells c o n t a i n e d in the c u l t u r e are i n d i c a t e d under abscissa. SPL: s p l e e n cells, G-10 SPL: m a c r o p h a g e - d e p r i v e d SPL, MP: m a c r o p h a g e s .
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~n u~g_/LO p r i m a r y a n t i - S R B C r e s p o n s e of m a c r o p h a g e - d e p r i v e d s p l e e n m e n t e d w i t h SA, NA, or SNB p e r i t o n e a l e x u d a t e cells. Indications as in Fig. 6.
cells s u p p l e are the same
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I" RESPONSE 0
(EXP.2)
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(EXP.3)
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FIG. 8 In v ~ O primary anti-SRBC response of macrophage-deprived spleen cells supplemented with SA, NA, or SNB macrophages. Indications are the same as in Fig. 6.
DISCUSSION Phagocytosis and stimulation of lymphocytes may be the two principal functions of macrophages, we are not sure, however, whether these two functions are always accompany each other. One hint is found in the investigation of Wiener and Bandieri (27) on the peritoneal macrophages of Biozzi mice. These mice comprise two selected lines with regard to nonspecific responsiveness in antibody responses (28). They reported that phagocytic activity by macrophages of low responder mice is higher than that of high responder mice. Relative to ontogeny of immune responsiveness, newborn mice can be regarded as low responders and adult mice as high responders. As we expected, A~cell activity of newborn (SNB) macrophages during the primary antibody response of adult spleen lymphocytes was much lower than that of adult (SA) macrophages (fig. 7 and Fig. 8). Phagocytic activity of SNB macrophages, however, was modestly higher than that of SA macrophages. It is not clear, however, whether such an antiparallelism between phagocytic activity and A-cell activity is applicable to normal macrophages in the peritoneal cavity and also in other tissues. Among normal macrophages, only adult peritoneal macrophages were studied in this experiment, since we could not obtain sufficient numbers of PEC from newborn mice. It may be necessary hereafter to study normal macrophages of newborn mice. Normal macrophages (NA) were more active than TGC-stimulated macrophages (SA and SNB) in not only phagocytosis of biologically inactive particles such as FE or latex (Fig. 3), but also special receptor-mediated phagocytosis of EA and EAC (Fig. 2 and Fig. 4). This did not agree with the experimental
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results of Bianco et al. (17), who reported that SA macrophages of Swiss mice were more active in phgocytosing EA and EAC than NA macrophages. NA macrophages could only attach but not ingest EAC, whereas SA macrophages exhibited both functions. We suppose that strain differences of mice might be one reason for such a discrepancy. Reconstitution of macrophage-deprived spleen cells with peritoneal macrophages was effective in supporting not only secondary responses (Fig. 6) but also primary responses (Fig. 7 and Fig. 8). Difference in A-cell activity was not observed among NA, SA, and SNB macrophages in secondary responses. In primary responses, however, SNB macrophages were incompetent as A-cells. This may disclose at least two aspects of macrophage function in antibody response: i) activation of unprimed lymphoid cells to initiate antibody response, and 2) presentation of antigens and promotion of lymphocyte viability. Both functions may be essential for primary responses, but secondary responses do not seem to necessarily require the first function. It was argued by Pierce and Kapp (29) that "lymphoid cells from immunnized animals are less dependent on some macrophage function for developement of antibody response in v~%o than are lymphoid cells from virgin mice". According to Erb and Feldmann (30), induction of helper T cells requires the aid of some factor(s) released from macrophages which may be different from factor(s) released from helper T cells for B cell activation on macrophages(31). MoreoVer, Katz and Unanue (32) reported that even antigen-bound fibroblasts were capable of stimulating primed lymphoid cells to elicit in v i ~ o secondary response. Thus, some special function of macrophages is required to initiate primary responses to thymus-dependent antigens, and it seems reasonable to assume that such a function is lacking in newborn macrophages at least in TGC-stimulated macrophages. Another possibility, however, which may not be completely excluded is that suppressor T cells are predominantly induced in response to stimuli from newborn macrophages. Mosier and Johnson (8) found that T cells in suckling mice act suppressively on responses of adult lymphoid cells to SRBC. If such suppressor T cells are formed in cooperation with macrophages, newborn macrophages may be understood as a suppressor inducer. This hypothesis remains to be investigated. We described in the introduction the still unsettled problem concerning which group of immune-participating cells is the most responsible for immunological immaturity of newborn animals. Perhaps B cells are not so responsible, since even one week-old mice can respond to thymus-independent antigens (8,9). Mosier and Johnson (8) and Fidler et al. (33) presented data which may indicate that lymphoid cells, but not macrophages, are responsible for immunlogical unresponsiveness of newborn mice. They found that one week (33) or two weeks (8)-old mouse spleen adherent cells could support the in vitro anti-SRBC primary response of adult spleen nonadherent cells, but combination of adult adherent cells with young mouse nonadherent cells did not result in antibody response. Landahl (34) reported, however, that A cell activity of newborn (3-9 days of age) spleen adherent cells was significantly recognizable but mich lower than that of adult adherent cells. Several investigators found that supplementation of newborn animals with adult peritoneal exudate cells resulted in the accerelation of immunological maturation (10-13). As we pointed out, their observations do not seem to
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present definite evidence that immaturity of macrophages should be a limiting factor in the ontogeny of immune responsiveness. What we can conclude from the present experiment is that TGC-stimulated peritoneal macrophages from newborn mice are incompetent, in contrast to those from adult mice, to support in v ~ 0 primary responses of macrophage-deprived spleen lymphoid cells, but such newborn macrophages are active in phagocytosing foreign materials. Thus, phagocytic activity and A-cell activity are not always parallel functions, and this condition in the developing mammal probably mimics the condition in phylogeny (35). ACKNOWLEDGMENTS We are grateful to Prof. E.L.Cooper, University of California at Los Angeles, for his helpful comments on this manuscript, and also to Prof. B.Cinader, University of Toronto, for his critical reading of the manuscript. REFERENCES V
i. STERZL, J. and SILVERSTEIN, A. M. Developmental aspects of immunity. Advances in Immunol. 6, 337, 1967. 2. SHORTMAN, K., DIENER, E., RUSSELL, P. and ARMSTRONG, W. D. The role of nonlymphoid accessory cells in the immune response to different antigens. J. Exp. Med. 131, 461, 1970. 3. GORCZYNSKI, R. M., MILLER, R. G. and PHILLIPS, R. A. In vivo requirement for a radiation-resistant cell in the immune response to sheep erythrocytes. J. Exp. Med. 134, 1201, 1971. 4. MOSIER, D. E. and COPPLESON, L. W. A three-cell interaction required for the induction of the primary immune response in vitro. Proc. Nat. Acad. Sci., U.S. 61, 542, 1968. 5. SPEAR, P. G., WANG, A-L., RUTISHAUSER, U. and E D E L ~ N , G . M . zation of splenic lymphoid cells in fetal and newborn mice. 138, 557, 1973.
CharacteriJ. Exp. Med.
6. SPEAR, P. G. and EDELMAN, G. M. Maturation of the humoral immune response in mice. J. Exp. Med. 139, 249, 1974. 7. GELFAND, M. C., ASOFSKY, R. and PAUL, W. E. Ontogeny of B lymphocytes. I. In vitro appearance of Ig-bearing lymphocytes. Cell. Immunol. 14, 460, 1974. 8. MOSIER, D. E. and JOHNSON, B. M. Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes. J. Exp. Med. 141, 216, 1975. 9. RABINOWITZ, S. G. Measurement and comparison of the proliferative and antibody responses of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens. Cell Immunol. 21, 201, 1976. i0. ARGYRIS, B. F. Role of macrophages in immunological maturation. Med. 128, 459, 1968. 11. BENDINELLI, M., SENESI, S. and FALCONE, G.
J. Exp.
Effect of adult peritoneal
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cells on the antibody response of newborn mice to sheep red blood cells. J. Immunol. 106, 1681, 1971. 12. MURCZYNSKA, W., ANDRZEJEWSKI, J. and BOGUNOWICZ, A. Role of macrophages in the development of immunological maturity in rat. Nature, 227, 721, 1970. 13. MARTIN, W. J. The cellular basis of immunological tolerance in newborn animals. Aust. J. Exp. Biol. Med. Sci. 44, 605, 1966. 14. HARDY, B., GLOBERSON, A. and DANON, D. Ontogenic development of the reactivity of macrophages to antigenic stimulation. Cell. Immunol. 9, 282, 1973. 15. METCHNIKOFF, E. Lectures on the Comparative Pathology of Inflammation. 1892. Reprinted by Dover Publications, Inc., New York, 1968. 16. REIF, A. E. and ALLEN, J. M. V. 209,521, 1966.
Mouse thymic iso-antigens.
Nature.
17. BIANCO, C., GRIFFIN, F. M. JR. and SILVERSTEIN, S. C. Studies of the macrophage complement receptor. Alteration of receptor function upon macrophage activation. J. Exp. Med. 141, 1278, 1975. 18. DODGE, J. T., MITCHELL, C. and HANAHAN, D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch. Biochem. Biophys. i00, 119, 1963. 19. THEOFILOPOULOS, A. N., DIXON, F. J. and BOKISCH, V. A. Binding of soluble immune complexes to human lymphoblastoid cells. I. Characterization of receptors for IgG Fc and complement and description of the binding mechanism. J. Exp. Med. 140, 877, 1974. 20. CHEN, C. and HIRSCH, J. G. The effects of mercaptoethanol and of peri toneal macrophages on the antibody-forming capacity of nonadherent mouse spleen cells in vitro. J. Exp. Med. 136, 604, 1972. 21. INABA, K., NAKANO, K. and MURAMATSU, S. Regulatory function of T lymphocytes in the immune response to polyvinyl pyrrolidone. I. Two categories of suppressor T cells. Cell. Immunol. in press. 22. ERB, P. and FELDMANN, M. The role of macrophages in the generation of T helper cells. I. The requirement for macrophages in helper cell induction and characteristics of the macrophage-T cell interaction. Cell. Immunol. 19, 356, 1975. 23. LY, I. A. and MISHELL, R. I. Separation of mouse spleen cells by passage through columns of Sephadex G-10. J. Immunol. Methods. 5, 239, 1974. 24. MARBROOK, J. Primary immune response in cultures of spleen cells. Lancet, 2, 1279, 1967. 25. JERNE, N. K., NORDIN, A. A. and HENRY, C. The agar plaque technique for recognizing antibody-producing cells. In Cell Bound Antibodies. (B. Amos and H. Koprowski, Ed.), Philadelphia: Wister Inst. Press, 1963, p. 109. 26. HOSONO, M. and MURAMATSU, S.
Use of 2-mercaptoethanol for distinguishing
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between IgM and IgG antibody-producing cells of mice immunized with bovine y globulin. J. Immunol. 109, 857, 1972. 27. WIENER, E. and BANDIERI, A. Differences in antigen handling by peritoneal macrophages from the Biozzi high and low responder lines of mice. Eur. J. Immunol. 4, 457, 1974. 28. STIFFEL, C., MOUTON, D., BOUTHILLIER, Y., HEUMANN, A. M., DECREUSFOND, C., MEVEL, J. C. AND BIOZZI, G. Polygenic regulation of general antibody synthesis in the mouse. Progress in !nununol. 11-2, 203, 1974. 29. PIERCE, C. W. and KAPP, J. H. The role of macrophages in antibody responses in vitro. In Immunobiology of the Macrophage. D. S. Nelson (Ed.) New York: Academic Press. 1976, p. i. 30. ERB, P. and FELDMANN, M. The role of macrophages in the generation of T helper cells. III. Influence of macrophage-derived factors in helper cell induction. Eur. J. Immunol. 5, 759, 1975. 31. FELDMANN, M. Cell interaction in the immune response in vitro. V. Specific collaboration via comple~es of antigen and thymus-derived cell immunoglobulin. J. Exp. Med. 136, 737, 1972. 32. KATZ, D. H. and UNANUE, E. R. Critical role of determinant presentation in the induction of specific response in immunocompetent lymphocytes. J. Exp. Med. 137, 967, 1973. 33. FIDLER, J. M., CHISCON, M. D. and GOLUB, E. S. Functional development of the interacting cells in the immune response. J. Immunol. 109, 136, 1972. 34. LANDAHL, C. A. Ontogeny of adherent cells. I. Distribution and ontogeny of A cells participating in the response to sheep erythrocytes in vitro. Eur. J. Immunol. 6, 130, 1976. 35. COOPER, E. L. Evolution of cell-mediated immunity. In Developmental Immunobiology. U. B. Solomon and J. H. Horton (Eds.) Amsterdam: Elsevier-North Holland. 1977, p. 456.
ONTOGENY I.
PHAGOCYTIC
NEWBORN
KOJI
NAKANO~,
AND
Vol. 2, pp. 505-518, 1978 Pergamon Press, Inc.
OF MACROPHAGE ACTIVITY
ADULT
TOMOHIDE
MOUSE
AND
FUNCTION A-CELL
ACTIVITY
PERITONEAL
HOSOKAWA§
AND
OF
MACROPHAGES*
SHIGERU
MURAMATSU¶
Department of Zoology, Faculty of Science, Kyoto University, Kitashirakawa-0iwakecho, Sakyo-ku, Kyoto, Japan 606
ABSTRACT. Phagocytic activity and immune-participating A-cell acitivity of newborn and adult mouse macrophages were investigated under in vi2AO conditions. Thioglycollate medium-stimulated newborn (SNB) or adult (SA), and nonstimulated adult (NA) peritoneal macrophages were used. Immune complexes of sheep erythrocytes (SRBC), aldehyde-fixed SRBC, and latex beads were employed in phagocytosis tests. A-cell activity was estimated as the capacity to support primary or secondary responses of macrophage-deprived adult spleen cells to SRBC. Results obtained were: i) phagocytic activity of NA macrophages was highest and that of SNB macrophages was higher than SA macrophages, 2) no difference was observed in A-cell acitivity for secondary response among SNB, SA, and NA macrophages, and 3) SNB macrophages were lacking in A-cell activity for primary response. These results suggest that the substantial A-cell function should be evaluated in primary responses, and that the A-cell and phagocytic functions do not necessarily accompany each other.
INTRODUCTION Newborns of mice and other animals are known to be immunologically immature (i). In adult animals, thymus-derived T lymphocytes, nonthymus-derived B lym-
* Supported by grant in aid for scientific research from the Ministry of Education, Science and Culture of Japan. Visiting investigator from the Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto. § Present address: Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kyoto. ¶ To whom correspondence should be sent. 01451305X17810701-05051502.0010 Copyright© 1978 Pergamon Press
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phocytes, and macrophages as accessory cells (A-cells) are involved in the immune responses (2,3). Interaction among T lymphocytes,B lymphocytes and A-cells are necessary for antibody responses to so-called thymus-dependent antigens (2-4). Thus, immunological unresponsiveness of newborn animals must be attributable to the immaturity of at least one or at most all of these three categories of cells. According to some authors, the ontogenetic differentiation of B cells may precede that of T cells (5-9). On the other hand, inoculation of adult peritoneal exudate cells (PEC) into newborn mice (i0,ii), rats (12) or rabbits (13) slightly but significantly enhanced the antibody response. In another experiment (14), only adult PEC, but not newborn PEC, were effective in restoring immune responsiveness of sublethally irradiated adult mice. These reports, however, do not seem to give evidence that the deficiency of macrophages in A-cell function is mainly responsible for immunological unresponsiveness of newborn animals, since lymphocytes may be contained in adult PEC preparations. Phagocytosis* of foreign materials is, undoubledly, the most fundamental and basic function of macrophages, a universal characteristic of the entire animal kingdom (15). By comparison, lymphocyte-stimulating A-cell activity seems to be relatively refined and more phylogenetically evolved function. Therefore, it may be problematical whether A-cell activity is directly related to phagocytic acitvity, or whether these two activities are intrinsically independent of each other. In this study, we investigated phagocytic activity and A-cell activity of peritoneal macrophages of newborn and adult mice. METHODS Mice : C3H/HeMs mice of both sexes (supplied from the Institute of Laboratory Animals, Faculty of Medicine, Kyoto University) were used. The age of adult mice in these experiments was about 2 months. AKR mice were used as a source of C5 -deficient serum and as anti-Thy 1.2 antibody producers. Peritoneal exudate cells (PEC) : Normal adult (NA), thioglycollate (TGC)-stimulated newborn (SNB), and adult (SA) mice were killed by exsanguination from the carotid artery and their peritoneal cavities flushed aseptically with Eagle's minimum essential medium (MEM) using plastic syringes. PEC were spun down at i~000 rpm for 5 minutes and resuspended in MEM with 10% fetal bovine serum (FBS, Flow Laboratories). TGC (Brewer's medium, Difco Laboratories) was injected intraperitoneally in the volume of 2 ml into adults or 0.2 ml into neonates (within one day after birth) 4 days before harvest of PEC. Preparation of macrophages for phagocytosis test : Glass petri dishes (60 mm diameter) including 8 rectangular glass cover slips (22 x i0 mm) on their bottoms were added with i x 106 PEC in the final volume of 5 ml, and kept for 22-24 hours at 370C in a C02 incubator (5% C0295% air). After then cover slips were washed thoroughly with MEM to remove nonadherent cells and FBS. Two cover slips each were transferred to 35 mm glass petri dishes containing 2 ml of MEM without FBS.
*
The term "phagocytosis" is used to include not only ingestion of test particles by macrophages, but also their attachment onto macrophage receptors.
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Erythrocytes : Sheep erythrocytes (E or SRBC) in Alsever's solution (Nikken Animal Blood Center, Kyoto) were washed three times with saline (0.15M NaCI) and suspended in MEM at 108 E/ml as the test particle preparation for phagocytosis test. Glutaraldehyde-fixed E (FE) and latex beads : Washed E were fixed with 1% glutaraldehyde, repeatedly washed and stored at 4°C in saline at 109 cells/ml until use. As test particles, FE were suspended in MEM at 108 cells/ml. Polystyrene latex beads (i.011 ~m diameter, Dow Chemical Co.) were suspended at a concentration of 0.1%. Antisera : Rabbit anti-E serum was obtained one week after intravenous injection of 2 x 109 E. IgG and IgM fractions of the antiserum were prepared by means of a Sephadex G-200 column. Anti-Thy 1.2 serum was prepared by immunizing AKR mice with C3H mouse thymocytes (16). Preparation of E-IgG complex (EA) : EA and EAC (see below) were prepared essentially according to the method of Bianco et al. (17). The i:i mixture of E suspension (109/ml) and anti-E IgG at a final concentration just below aggulutinating E was incubated with gentle shaking at 37°C for 15 minutes. After washing three times with MEM, EA thus prepared was suspended at 108 cells/ml. Preparation of E-IgM-C3 (EAC) and ghost EAC (GEAC) : E-IgM complex was prepared similarly to EA, and suspended at 109/ml in veronal-buffered saline containing glucose, Ca+2, Mg+2 and gelatin. Mixture at i:i of the E-IgM and i/i0 diluted fresh AKR serum was incubated at 37°C for i0 minutes. EAC thus prepared was washed three times with MEM and suspended at 108 cells/ml. E-IgM and AKR serum-treated E were also prepared as control test particles. Neither attachment nor ingestion of these particles was observed. GEAC was used for blocking C3 receptors on macrophages. Washed E were lysed in hypotonic phosphate buffer solution (18) and hemoglobin-free ghosts were obtained by repeated washing with the buffer by centrifugation at 15,000 rpm for 30 minutes. Ghosts were homogenized by a supersonicator, sedimented by centrifugation at 30,000 rpm, and resuspended in MEM at the concentration equivalent to 2 x 10 I° E/ml. Then, GEAC was prepared following the procedure similar to that for EAC, washed three times with MEM by centrifugation, and diluted to the concentration equivalent to 2 x 109 E/ml. Aggregated human gamma globulin emulsion (aHGG) : Human gamma globulin (Fraction II, Wako Purechemical Industry, Osaka) at a concentration of 50 mg/ml in saline was heated at 63°C for 20 minutes and centrifuged at 3,000 rpm for 30 minutes. Unsedimented fraction of aHGG was used for blocking Fc receptors on macrophages. Assay system for phagocytic activity : To each 35 mm glass petri dish containing two cover slips with macrophages and 2 ml of MEM without FBS was added 0.2 ml of one of the test particle preparations, and incubated for 120 minutes (see Fig. 2). Usually the dishes were incubated at 4°C for the attachment of particles onto macrophage receptors without ingestion, and at 37°C for the ingestion of particles. After incubation, cover slips were washed several times with shaking and fixed with 2% gutaraldehyde in MEM. After washing in distilled water, cover slips were reversed upside down, and embedded on slides with glycerin jelly. Macrophages were observed by negative phase contrast microscopy. To examine the ingestion
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of EA and EAC by macrophages, cover slips were dipped into 0.83% NH~CI for 5 minutes to remove extracellularly attached erythrocytes before fixation. In each test, at least 400 macrophages were exmined. The data concerning phagocytosis are presented in this paper according to Bianco et al. (17). Percent ingestion or attachment is the percentage of macrophages which ingested or attached test particles. Ingestion or attachment index is the number of particles ingested ar attached per i00 macrophageso The attachment was regarded as positive when one macrophages attached three or more EA or EAC. Experiments were repeated at lest three times for each preparation of test particles, and representative results are shown in this paper. Blockade of Fc receptors by aHGG and C3 receptors by GEAC : Cover slips with macrophages in 35 mm petri dishes were preincubated in 2 ml of MEM containing lO mg of aHGG or 0.6 ml of GEAC at 37°C for 30 minutes, and then test particles were added without changing the medium. Trypsin treatment of macrophages : To destroy selectively C3 receptors (17), cover slips with macrophages were incubated at 37°C for 20 minutes with 1 mg/ml of trypsin (Sigma Chemical Co.) in MEM. Cover slips were washed twice with i mg/ml of soybean trypsin inhibitor (Worthington Biochemical Co.) in MEM to terminate trypsin digestion, and submitted to the phagocytosis test in fresh MEM. Test for the A-cell activity in anti-SRBC response in vitro : A-cell activity of macrophages was assessed by the capacity to restore the in vig~o anti-SRBC response of macrophage-deprived spleen cells as described below. Culture medium was MEM with 2 mM L-glutamine and 10% FBS. In the case of dish culture, 5 x 10-SM 2-mercaptoethanol was added to the medium (20). Viability of tells was determined by the trypan blue dye exclusion test. PREPARATION OF LYMPHOCYTE-DEPLETED MACROPHAGES. We depleted lymphocytes from PEC by X-irradiation and anti-Thy 1.2 treatment without detriolating A-cell activity of macrophages (3,22). Normal and TGC-stimulated mice received whole body X-irradiation at 1,300 R with a therapeutic X-ray emitter (Toshiba Electric CoL, Tokyo). Irradiation was performed at 180 kV and 20 mA, target distance 44 cm, with 0.5 mm Cu and 0.5 mm AI filter, at the rate of i00 R/min. After irradiation, peritoneal cells were collected and treated with anti-Thy 1.2 serum and complement (guinea pig serum absorbed on C3H mouse spleen cells) (21), washed, and resuspended in the culture medium. PREPARATION OF MACROPHAGE-DEPRIVED SPLEEN CELLS. Normal or primed spleen cells were passed through Sephadex G-10 (Pharmacia Fine Chemicals) column according to the method of Ly and Mishell (23). Primed spleen cells were obtained from mice injected intravenously with 107 SRBC 4 days previously.
IN VITRO ANTI-SRBC RESPONSE. Marbrook's flasks (24) (Takahashi Giken, Tokyo) and plastic dishes (Falcon 3001, diameter 35 mm, Falcon, Oxnard) were employed. In primary responses, only Marbrook's flaskswere used. The test system usually contained 107 macrophage-deprived spleen cells, 5 x 106 SRBC, and 104-106 macrophages. Volumes of the culture medium were i ml in the inner chamber and i0 ml in the outer chamber of Marbrook's flasks, and 3 ml in plastic dishes. Cultures were maintained at 37°C in 5% C02 and 95% air without rocking for 4 days to obtain peak responses. Antibody-forming cells were assessed by the plaque-forming cell assay (25) in agarose gel on slides (26). Only direct plaque-forming cells (PFC) were detected in these experiments. Results are presented as the mean number of duplicate cultures.
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RESULTS Determination of optimum timing for collecting peritoneal macrophages for the phagocytosis test after TGC injection : PEC were collected on various days after injection of TGC into adult mice, cultured for 24 hours, and submitted to phagocytosis test using EA as test particles. As shown in Fig. i, both attachment and ingestion indices were highest in normal macrophages (day 0) and they fell off rapidly i day after TGC injection. The indices tended to increase again up to the gentle peak on day 3 to day 5. Thus, we decided to collect PEC 4 days after TGC injection as the source of SA and also SNB macrophages.
EA N
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1400
1200
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FIG. I Phagocytic activity of adult peritoneal macrophages collected on various days after TGC injection. Macrophages cellected on day 0 were TGC-unstimulated cells. Open and closed circles indicate, respectively, attachment and ingestion indices for EA. Figures in parentheses are percentages of macrophages carrying EA.
Kinetics of phagocytosis after addition of test particles : SA and NA macrophages were given EA and EAC at 4°C or at 37°C. At various times thereafter, attachment and ingestion of test particles were evaluated. As depicted in Fig. 2, both attachment and ingestion indices tended to increase until 2 hours following the addition of test particles. Thus, the phagocytosis test was performed for 2 hours in the following experiments. Phagocytic activities : Fig. 3 shows the ingestion of E, FE, and latex beads by NA, SA, and SNB macrophages. FE and latex were actively ingested at 37°C but the phagocy~osis of E was practically nil in any macrophage preparation. Ingestion indices for FE and latex were highest in NA, second in SNB, and lowest in SA macrophages. Attachment of these three kinds of particles on macrophages did not occur either at 37°C or at 40C. Results of experiments to investigate the phagocytic activity for EA and EAC are shown in Fig. 4. As in the case of FE and latex, NA macrophages were more phagocytic than the other two macrophage preparations, especially in the phagocytosis of EAC. In the data shown in Fig. 4, attachment and ingestion indices of NA macrophages were more than eight times and two times, respectively, as much as those of SA macrophages. Percentages of NA macrophages attaching or ingesting EAC were also remarkably higher than
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ONTOGENY OF h~CROPHAGE FUNCTION
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those of SA macrophages. Difference between SNB and SA macrophages was not so conspicuous, but any values of indices of SA macrophages did not surpass those of SNB macrophages.
1500
ATTACHMENT ( ) : %
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Time course of the phagocytic activity after initiation of SA or NA culture with EA or EAC. Open and closed symbols indicate, respectively, attachment (upper panel) and ingestion (lower panel) indices for EA or EAC. Circles and squares represent, respectively, indices of SA and NA macrophages. Figures in parentheses are percentages of macrophages carrying EA or EAC.
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INCUBATIONTIME (MIN)
Effect of aHGG~ GEAC and trypsin on attachment and ingestion EA and EAC : To confirm that EA was phagocytosed via Fc receptors on macrophages, blocking of receptors with aHGG was attempted before the addition of EA. Similarly, preincubation of macrophages with GEAC and pretreatment with trypsin were performed to block or digest C3 receptors. As shown in Fig. 5, both attachment and ingestion of EA were inhibited only by aHGG but not by GEAC or trypsin, and those of EAC were inhibited by GEAC or trypsin but not by aHGG. These results seem to indicate that EA and EAC were phagocytosed via different receptors, probably Fc receptors for EA and C3 receptors for EAC. Ingestion of latex particles was not affected by GEAC nor trypsin, but the ingestion index was somewhat reduced by aHGG. This does not seem, however, to be caused by masking of Fc receptors with aHGG, since percentage of macrophages ingesting latex beads was not reduced, as contrasted with the remarkable reduction of EA ingestion not only in the ingestion index but also in the percentage. A-cell activity in A-cell activity capacity to support cells. Experiments
secondary response : of macrophages in secondary response was estimated by their in v a ~ O antibody response of SRBC-primed spleen lymphoid were repeated five times and similar results were obtained,
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ONTOGENY OF MACROPHAGEFUNCTION
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C3H 1400
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[]
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CONTROL
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Effects of the pretreatment of macrophages with aHGG, trypsin, or GEAC on the attachment at 4°C or at 37°C (upper panel) and ingestion at 37°C (lower panel) of EA, EAC, or latex beads. NA macrophages were used in this experiment. Procedures in detail are in the text. Height of each column represents attachment or ingestion index, and figures on columns are percentages of macrophages carrying test particles.
so that the representative result is shown in Fig. 6. It is indicated that deprivation of macrophages from spleen cell preparation resulted in reduction in the number of PFC when compared with original cell preparations; any one preparation of lymphocyte-depleted macrophages effectively supported the antibody response of such primed lymphocytes. In dish cultures, the number of added macrophages was changed from i0 ~ to 106 , but no dose effect was observed. In a preliminary experiment, when only macrophages were cultured with antigen, no PFC were detected (data not shown). A~cell activity in primary response : Unprimed spleen cells deprived of macrophages were used to estimate A-cell activity of macrophages in primary in vi~YLo responses. In the experiment of Fig. 7, unirradiated and anti-Thy 1.2-untreated PFC were used. Lymphocytedepleted macrophages were used in the experiments of Fig. 8. In both Fig. 7 and Fig. 8, adult 105 and 106 macrophages (SA and NA) were able to reconstitute partly or completely i0 ? macrophage-deprived spleen cells effecting responses to SRBC. In experiment 3 (Fig. 8), an excess of SA macrophages (106 ) was ineefective in supporting the response. It should be noticed that A-cell activity of newborn macrophages (SNB) was very low in comparison with adult macrophages. This was the case both with lymphocyte-depleted macrophages (Fig. 8) and with the original PEC (Fig. 7).
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2 ° RESPONSE (£xP.1)
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In V~;C~LO s e c o n d a r y a n t i - S R B C r e s p o n s e of m a c r o p h a g e - d e p r i v e d s p l e e n cells suppl e m e n t e d w i t h SA, NA, or SNB m a c r o p h a g e s . H e i g h t of each c o l u m n refers to the n u m b e r of PFC per c u l t u r e (the g e o m e t r i c a l m e a n of d u p l i c a t e cultures) in p l a s t i c dish (left panel) or M a r b r o o k ' s f l a s k (right panel). O p e n c i r c l e s rep r e s e n t the a r i t h m e t r i c a l m e a n of d u p l i c a t e assays for each culture. Sources and n u m b e r s of cells c o n t a i n e d in the c u l t u r e are i n d i c a t e d under abscissa. SPL: s p l e e n cells, G-10 SPL: m a c r o p h a g e - d e p r i v e d SPL, MP: m a c r o p h a g e s .
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~n u~g_/LO p r i m a r y a n t i - S R B C r e s p o n s e of m a c r o p h a g e - d e p r i v e d s p l e e n m e n t e d w i t h SA, NA, or SNB p e r i t o n e a l e x u d a t e cells. Indications as in Fig. 6.
cells s u p p l e are the same
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I" RESPONSE 0
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FIG. 8 In v ~ O primary anti-SRBC response of macrophage-deprived spleen cells supplemented with SA, NA, or SNB macrophages. Indications are the same as in Fig. 6.
DISCUSSION Phagocytosis and stimulation of lymphocytes may be the two principal functions of macrophages, we are not sure, however, whether these two functions are always accompany each other. One hint is found in the investigation of Wiener and Bandieri (27) on the peritoneal macrophages of Biozzi mice. These mice comprise two selected lines with regard to nonspecific responsiveness in antibody responses (28). They reported that phagocytic activity by macrophages of low responder mice is higher than that of high responder mice. Relative to ontogeny of immune responsiveness, newborn mice can be regarded as low responders and adult mice as high responders. As we expected, A~cell activity of newborn (SNB) macrophages during the primary antibody response of adult spleen lymphocytes was much lower than that of adult (SA) macrophages (fig. 7 and Fig. 8). Phagocytic activity of SNB macrophages, however, was modestly higher than that of SA macrophages. It is not clear, however, whether such an antiparallelism between phagocytic activity and A-cell activity is applicable to normal macrophages in the peritoneal cavity and also in other tissues. Among normal macrophages, only adult peritoneal macrophages were studied in this experiment, since we could not obtain sufficient numbers of PEC from newborn mice. It may be necessary hereafter to study normal macrophages of newborn mice. Normal macrophages (NA) were more active than TGC-stimulated macrophages (SA and SNB) in not only phagocytosis of biologically inactive particles such as FE or latex (Fig. 3), but also special receptor-mediated phagocytosis of EA and EAC (Fig. 2 and Fig. 4). This did not agree with the experimental
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results of Bianco et al. (17), who reported that SA macrophages of Swiss mice were more active in phgocytosing EA and EAC than NA macrophages. NA macrophages could only attach but not ingest EAC, whereas SA macrophages exhibited both functions. We suppose that strain differences of mice might be one reason for such a discrepancy. Reconstitution of macrophage-deprived spleen cells with peritoneal macrophages was effective in supporting not only secondary responses (Fig. 6) but also primary responses (Fig. 7 and Fig. 8). Difference in A-cell activity was not observed among NA, SA, and SNB macrophages in secondary responses. In primary responses, however, SNB macrophages were incompetent as A-cells. This may disclose at least two aspects of macrophage function in antibody response: i) activation of unprimed lymphoid cells to initiate antibody response, and 2) presentation of antigens and promotion of lymphocyte viability. Both functions may be essential for primary responses, but secondary responses do not seem to necessarily require the first function. It was argued by Pierce and Kapp (29) that "lymphoid cells from immunnized animals are less dependent on some macrophage function for developement of antibody response in v~%o than are lymphoid cells from virgin mice". According to Erb and Feldmann (30), induction of helper T cells requires the aid of some factor(s) released from macrophages which may be different from factor(s) released from helper T cells for B cell activation on macrophages(31). MoreoVer, Katz and Unanue (32) reported that even antigen-bound fibroblasts were capable of stimulating primed lymphoid cells to elicit in v i ~ o secondary response. Thus, some special function of macrophages is required to initiate primary responses to thymus-dependent antigens, and it seems reasonable to assume that such a function is lacking in newborn macrophages at least in TGC-stimulated macrophages. Another possibility, however, which may not be completely excluded is that suppressor T cells are predominantly induced in response to stimuli from newborn macrophages. Mosier and Johnson (8) found that T cells in suckling mice act suppressively on responses of adult lymphoid cells to SRBC. If such suppressor T cells are formed in cooperation with macrophages, newborn macrophages may be understood as a suppressor inducer. This hypothesis remains to be investigated. We described in the introduction the still unsettled problem concerning which group of immune-participating cells is the most responsible for immunological immaturity of newborn animals. Perhaps B cells are not so responsible, since even one week-old mice can respond to thymus-independent antigens (8,9). Mosier and Johnson (8) and Fidler et al. (33) presented data which may indicate that lymphoid cells, but not macrophages, are responsible for immunlogical unresponsiveness of newborn mice. They found that one week (33) or two weeks (8)-old mouse spleen adherent cells could support the in vitro anti-SRBC primary response of adult spleen nonadherent cells, but combination of adult adherent cells with young mouse nonadherent cells did not result in antibody response. Landahl (34) reported, however, that A cell activity of newborn (3-9 days of age) spleen adherent cells was significantly recognizable but mich lower than that of adult adherent cells. Several investigators found that supplementation of newborn animals with adult peritoneal exudate cells resulted in the accerelation of immunological maturation (10-13). As we pointed out, their observations do not seem to
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present definite evidence that immaturity of macrophages should be a limiting factor in the ontogeny of immune responsiveness. What we can conclude from the present experiment is that TGC-stimulated peritoneal macrophages from newborn mice are incompetent, in contrast to those from adult mice, to support in v ~ 0 primary responses of macrophage-deprived spleen lymphoid cells, but such newborn macrophages are active in phagocytosing foreign materials. Thus, phagocytic activity and A-cell activity are not always parallel functions, and this condition in the developing mammal probably mimics the condition in phylogeny (35). ACKNOWLEDGMENTS We are grateful to Prof. E.L.Cooper, University of California at Los Angeles, for his helpful comments on this manuscript, and also to Prof. B.Cinader, University of Toronto, for his critical reading of the manuscript. REFERENCES V
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