INFECTION AND IMMUNITY, Dec. 1979, 0019-9567/79/12-1 129/08$02.00/0

p. 1129-1136

Vol. 26, No. 3

Comparison of Peritoneal Macrophages from Germfree and Conventional Mice BERIT M0RLAND,* ALF I. SMIEVOLL, AND TORE MIDTVEDTt The Institute of Medical Biology, University of Troms0, Troms0, Norway Received for publication 24 May 1979

Morphology, lysosomal enzyme activities, and phagocytosis via immunological receptors were tested in peritoneal macrophages from germfree and conventional mice. Nonstimulated macrophages from germfree mice showed less spreading and were more easily detached when seeded on glass than conventional macrophages. The activities of the lysosomal acid phosphatase and cathepsin D were similar in the two cell groups, whereas 83-glucuronidase showed higher activity in macrophages from germfree mice. F. receptor-mediated phagocytosis of opsonized sheep erythrocytes was equally effective in germfree and conventional macrophages, and both cell types attached but did not internalize erythrocytes via the C:3b receptor. Intraperitoneal injections of mineral oil caused a significantly higher influx of macrophages in conventional mice than in germfree mice, whereas the influx of polymorphonuclear cells was enhanced in both animals. Stimulation in vivo with oil or Escherichia coli endotoxin increased cell size, spreading ability, membrane ruffling, and lysosomal enzyme activities in macrophages from both conventional and germfree mice. The Fc-mediated phagocytosis was not influenced by stimulation, whereas the capacity to internalize via C3b receptor was triggered in macrophages from conventional mice, but not in corresponding cells from germfree mice. Similar results were obtained after stimulation with endotoxin in vitro. Culture in fetal calf serum for 72 h caused intracellular rises in all three enzyme activities tested in macrophages from conventional mice, whereas only the activity of acid phosphatase was increased in macrophages from germfree mice. Stimulation with zymosan in vitro caused selective release of lysosomal enzyme activity in macrophages from both animal groups. We conclude that peritoneal macrophages from germfree mice share several properties with cells from conventional mice, however, unstimulated 83-glucuronidase activity was increased, whereas spreading on glass, chemotactic response, in vitro induction of lysosomal enzymes, and the capacity to internalize via the C3b receptor after stimulation were reduced or absent. The lymphatic tissues of germfree (GF) animals have been shown to respond to antigenic stimulation qualitatively similarly to conventional (CONV) animals, although the reactions may differ in rate and intensity (4, 13, 20, 34). GF animals are able to synthesize immunoglobulins (12, 34), and their rejection of skin allografts is similar to that of CONV animals (32). Generation of lymphocyte-dependent, antigeninduced monocyte chemotactic activity is also normal in GF animals (14). However, GF animals are less resistant to tubercle bacilli and other infectious agents that provoke a vigorous cell-mediated immune response in CONV subjects (35; E. M. Lerner, Fed. Proc. 23:286, 1964). t Present address: Kaptein W. Wilhelmsen og, Frues Bakteriologiske Institutt, National Hospital of Norway, Oslo, Norway.

According to most authors, they also fail to express, or show only feeble, delayed-type hypersensitivity (DTH) reactions to antigens that elicit marked responses in CONV animals (16, 36; Lerner, Fed. Proc. 23:286, 1964). It has long been known that macrophages are essential for the expression of DTH and cellular resistance to infection (18, 24). It is therefore logical to ask whether the immune deficiency in GF animals is related to a defect in macrophages. Meltzer found that macrophages from GF nude mice were not tumoricidal in vitro, whereas corresponding cells from CONV nude mice were cytotoxic to tumor cells in a way similar to Mycobacterium bovis BCG-stimulated macrophages from CONV normal mice (19). Macrophages from GF animals respond poorly to chemotactic stimuli that attract the macro-

1129

1130

MORLAND, SMIEVOLL, AND MIDTVEDT

phages of CONV subjects, and there is a lower influx of monocytes into the peritoneal cavity after peptone injections in GF mice compared with CONV mice (14). Kupffer cells (5) and alveolar macrophages (E. R. Heise and Q. Myrvik, Fed. Proc. 25:439, 1966) derived from GF rats show lower lysosomal enzyme activities than are found in corresponding cells from CONV rats. The present work was undertaken to study these and other macrophage functions of importance in the immune defense system (7). Peritoneal macrophages from GF mice were used in the study, since these cells are well characterized in the conventional state (8, 23). Numerous reports on CONV animals have shown that the metabolic state of macrophages changes after exposure to bacteria and bacterial products; the cells become activated (18, 24). Even subclinical infections obtained through maintenance in conventional animal houses greatly influence macrophage activities (11). Another aspect of this work was therefore to gain more insight into macrophage activation by studying properties of macrophages without any previous bacterial contact and their response to activation (25). MATERIALS AND METHODS Animals. Mice of the NMRI strain, 5 to 6 weeks old and weighing about 20 g, were used. The GF mice were raised and reared as described earlier (21). All experiments started within 1 h after the GF mice were taken out of the isolators. Serum deficient in C5 was prepared from AKR mice. Macrophage cultures. Harvesting and culture methods were essentially those described by Cohn and Benson (8). Cells were counted in Burker hemocytometer after methyl violet staining (Chroma-Gesellschaft, Stuttgart, West Germany) and seeded to give 106 cells/ ml in minimal essential medium (Eagle) with Earle salts (MEM) (GIBCO Laboratories, Grand Island, N.Y.) with 20% heat-inactivated (560C, 30 min) fetal calf serum (FCS) (GIBCO), 100 IU of penicillin per ml, and 100 ytg of streptomycin per ml. The cells were cultured in plates (Linbro Chemical Co., New Haven, Conn.) with glass cover slips (14-mm diameter) in 5% CO2 in air at 370C. After 2 h the cultures were washed with medium (370C) to remove nonadherent cells, and new medium was added. At 6 h the cultures were either used for experimentation or supplied with new medium and cultured for various periods, with or without stimulants (see below). Stimulating agents. In vivo stimulation of macrophages was achieved by injecting GF or CONV mice intraperitoneally (i.p.), 3 days before collecting the peritoneal exudates, with either lyophilized endotoxin (Escherichia coli 026:B6, Boivin preparation; Difco Laboratories, Detroit, Mich.) suspended in sterile saline, 70 tyg per mouse (23), or mineral oil, 1 ml per mouse (23). The GF mice were kept under sterile conditions during this period. Neither of the injected agents caused any reduction in animal viability (1).

INFECT. IMMUN. In vitro stimulation of macrophages was performed by adding to resident GF or CONV cultures either endotoxin, 10 fig per ml of medium (23), or zymosan (Sigma Chemical Co., St. Louis, Mo.) boiled in sterile saline, 10 jg per ml of medium (29). Preparation of media and cell lysates for biochemical determinations. At the times indicated, the media were collected, centrifuged to remove cell debris, and stored frozen (-70°C) until use. The cell monolayers were frozen and thawed once in Triton X100 (BDH Chemicals, Ltd., Poole, England) (0.1% [vol/voll in distilled water) (enzyme assays) or 0.1 N NaOH (protein assay) before analysis. Biochemical determinations. Acid phosphatase (EC 3.1.3.2), cathepsin D (EC 3.4.4.23), and fB-glucuronidase (EC 3.2.1.31) were measured as described by Barrett (3, 23). Enzyme activities were expressed in milliunits, 1 U being the amount of enzyme necessary to release 1 [Lmol of product per min. All reagents for lysosomal enzyme determinations were from Sigma. Lactate dehydrogenase (LDH) (EC 1.1.1.27) was assayed by a kit from Boehringer (Mannheim GmbH, West Germany). Protein in the lysates was determined according to the method of Lowry et al. (17), with bovine serum albumin (Sigma) as standard. Phase-contrast microscopy. The cell monolayers were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) with 0.1 M sucrose. Cover slips were mounted on a drop of Aquamont (Edward Gurr Ltd., London). The slides were examined with a Zeiss phasecontrast photomicroscope. Kodak Tri-X pan blackand-white film was used for photography. Phagocytosis studies. The test particles used for phagocytosis studies were sheep erythrocytes (E) (National Institute of Health, Oslo, Norway) opsonized with either rabbit anti-sheep erythrocyte immunoglobulin G [E(IgG); Cordis Laboratories, Miami, Fla.], or IgM (Cordis) plus complement factors in C5deficient mouse serum [E(IgM)C] as described previously (23). For attachment to macrophage surfaces, the macrophage monolayers were incubated with the erythrocytes at 4°C for 30 min. Controls containing erythrocytes E(IgM) did not attach to the macrophages. The cultures were washed, and some were kept to calculate the attachment. Others were transferred to 37°C for 60 min to perform internalization of erythrocytes. At the end of the incubation period, the monolayers were treated with a 0.14 M solution of NH4Cl (37°C) for 2 min to lyse attached, noninternalized erythrocytes and washed. The results were expressed as the percentage of macrophages that had attached or internalized three or more erythrocytes, determined by phase-contrast microscopy of at least 500 macrophages on each cover slip. Statistics. The results of biochemical determinations were subjected to the Wilcoxon paired-comparison test, and a-values below 0.05 were considered to accompany statistically significant differences. All results are given as mean ± standard errors of the mean.

RESULTS Peritoneal cell number. There were no differences in resident peritoneal cell counts be-

VOL. 26, 1979

MACROPHAGE FUNCTION IN GERMFREE MICE

1131

tween GF and CONV mice (Table 1). The periInjection i.p. of endotoxin did not affect total toneal exudate obtained from each mouse con- cell number or the percentage of macrophages tained 2 x 106 to 5 X 106 cells, of which 50% in the peritoneal exudate from both GF and showed macrophage characteristics. CONV mice (Table 1). Injection i.p. of mineral oil caused an increase of the total peritoneal cell TABLE 1. Total cell number of peritoneal exudates" number in CONV mice, and about 80% of these cells showed macrophage characteristics (Table from CONV and GF mice, without or after 1). Oil injections in GF mice caused a small rise stimulation in vivo in total cell count of the peritoneal exudate Peritoneal cells/mouse (x 106) (Table 1). In this case, however, an increased In vivo stimulus CONV GF number of polymorphonuclear granulocytes acfor the increase in total cell number. counted ± 0.3 2.8 0.3 3.3 ± None Macrophage morphology. When examined 2.4 ± 0.4 Endotoxin 3.3 ± 0.6 after 6 h of culture, resident macrophages from 10.4 ± 5.3b 4.7 ± 0.4b Oil mice were spread out, with long, thin "The cells in freshly harvested exudates were CONV extensions radiating from the cells (Fig. la). The stained with methyl violet and counted in Burcker hemocytometer. Means of five to seven experiments resident macrophages from GF mice were more rounded, with few extensions (Fig. lb). These ± standard error. b a < 0.05 compared with corresponding cells with- cells could more easily be detached from the out stimulus. cover slips than cells from CONV mice.

FIG. 1. Phase-contrast (X665) micrographs of CONV and GF macrophages cultured for 6 h (a to c) and 72 h (d). (a) Nonstimulated CONV macrophages; (b) nonstimulated GF macrophages; (c) in vivo endotoxinstimulated GF macrophages; (d) in vitro MEM + 20%c/r FCS-cultured GF macrophages.

1132

M0RLAND, SMIEVOLL, AND MIDTVEDT

The difference between cells from GF and CONV mice disappeared after in vivo stimulation. Thus, macrophages obtained from endotoxin-treated CONV as well as GF mice were larger and more spread out than the corresponding resident cells. Membrane ruffling was observed in both cell types. Endotoxin-stimulated GF cells are shown in Fig. ic. Macrophages from oil-treated CONV and GF animals were intermediate between resident and endotoxin-stimulated macrophages with respect to spreading and ruffling (not shown). Cultivation in FCS-supplemented medium caused spreading of the resident GF macrophages, and after 72 h in vitro the initial differences between resident GF and CONV macrophages had almost disappeared (Fig. 1d). Addition of endotoxin or zymosan in vitro caused further enlargement of both cell types, and endotoxin-treated macrophages from GF as well as CONV mice showed membrane ruffling similar to what was observed after endotoxin stimulation in vivo (not shown). Enzyme activities in cells and media. Table 2 shows the specific activities of three lysosomal enzymes tested in lysates of GF and CONV macrophages after 6 h in vitro. The activities of acid phosphatase and cathepsin D were similar in the two cell groups, whereas ,8glucuronidase activity was higher in lysates obtained from GF macrophages. In vivo treatment with endotoxin caused a marked rise in acid phosphatase activity in both cell groups (Fig. 2a) and no change of cathepsin D activity (Fig. 2b), whereas there was a moderate increase in f-glucuronidase activity (Fig. 2c). Injection i.p. of oil caused rises in all enzyme activities tested (Fig. 2a-c). All increases were similar in GF and CONV macrophages. Stimulation in vitro by FCS (20%, 72 h) increased the intracellular activities of all three enzymes tested in CONV macrophages (Fig. 3). The activity of acid phosphatase only was increased in GF macrophages under the same conditions (Fig. 3a), whereas fl-glucuronidase and cathepsin D stayed at the 6 h level or decreased with time in culture (Fig. 3b, c). When TABLE 2. Lysosomal enzyme activities' in macrophages from CONV and GF mice Macrophage type

Enzyme activity (mU/mg of protein) Acid phos-

Cathepsin D /3-Glucuroniphatase dase CONV ... 2.06 + 0.05 106.0 ± 26.0 1.42 ± 0.21 GF ... 1.88 ± 0.49 86.2 ± 21.9 2.52 ± 0.49h "Assayed at 6 h in vitro. Means of five experiments ± standard error. ^a < 0.05 compared with CONV macrophages.

INFECT. IMMUN %

500 -

x)

400-

x)

I-

x) I

x)

300200-

100E

N

E

0

00

%'4

300 200 -

x)

X)

-I-T

X)

100N

E

0

CONV macrophages

E

GF macrophages

FIG. 2. Effect of in vivo stimulation with endotoxin (E) or mineral oil (0) on lysosomal enzyme activities

in CONV and GF macrophages. Enzyme activities are calculated as milliunits per milligram of protein and expressed as percentage of corresponding activ-

ities in nonstimulated cells (N). Mean + standard of five separate experiments. x), a < 0.05 compared with N. (a) Acid phosphatase; (b) cathepsin D; (c) ,8-glucuronidase. error

endotoxin was added together with FCS, a further increase of acid phosphatase activity was found in CONV as well as GF macrophages (Fig. 3a). The activity of 8-glucuronidase was assayed in the media from the various cultures, together with LDH to estimate the percentage of enzyme release due to cell damage. Between 10 and 20% of the total activity of both enzymes was detected in all media after 24 and 48 h of cultivation in FCS (Fig. 4) or FCS plus endotoxin (not shown). When CONV and GF macrophages were cultured in the presence of 20% FCS plus 10 tig of zymosan per ml, about 45% of total /8glucuronidase activity, and only about 20% of total LDH activity, was detected in the media of

VOL. 26, 1979

MACROPHAGE FUNCTION IN GERMFREE MICE

1133

I

1400 1300 12001 1001000

900800

x)

I

91

700*

'x)

600 500 400 300 200100

E

50-

-, 40E 30. , 20,10-

G)

x)

6

FCS FCS

FCS FCS E

E

24

48

Time(hours)

4J

>8

%

(U N

%*

200-

100-

FC{S11FjX

FCS

N

C 50L 40-

E

.~~~~~~I

3019

20106

N CONV macrophages 6

hours

GF macrophages 72

hours

FIG. 3. Effect of 72 h of culture in MEM + 20% FCS (FCS) or MEM + 20%o FCS + endotoxin (10 jig! ml) (FCS + E) on lysosomal enzyme activities in CONV and GF macrophages. Enzyme activities are calculated as milliunits per milligram ofprotein and expressed as percentage of 6-h activities in nonstimulated cells (N). Mean ± standard error of five separate experiments. x), a < 0.05 compared with N; x), a < 0.05 compared with FCS. (a) Acid phosphatase; (b) cathepsin D; (c) ,8-glucuronidase.

both macrophage groups after 24 and 48 h of culture (Fig. 4). Phagocytosis via immunological receptors. The amount of opsonized sheep erythrocytes attached or internalized by means of Fc and C3b receptors is recorded in Tables 3 and 4. At 6 h about 95% of the resident CONV macrophages showed both attachment and internalization via the Fc receptor (Table 4). The percentage of CONV macrophages which attached particles by the C3b receptor was also very high, but less than 3% of the cells showed internalization of E(IgM)C (Table 3). Efficient attachment to both receptors as well as internalization via the Fc receptor were also found in GF resi-

24

48

Time(hours)

FIG. 4. Effect of 48 h of culture in MEM + 20% FCS (A) or MEM + 20%o FCS + zymosan (10 pg/ml) (0) on release of enzyme activities from CONV (a) and GF (b) macrophages. Media activities of 13-glucuronidase ( ) and LDH (--- ) are expressed as percentage of total culture activities. Mean ± standard offour separate experiments.

dent macrophages at 6 h (Table 3). However, the average number of erythrocytes attached or internalized was lower per resident GF macrophage (5 to 10 erythrocytes per macrophage) than per corresponding CONV macrophage (15 to 20 erythrocytes per macrophage). Like CONV macrophages, the resident GF macrophages did not internalize opsonized sheep erythrocytes via the C3b receptor (Table 3). Endotoxin or oil stimulation in vivo increased the number of opsonized particles which attached to Fc or C3b receptors of CONV as well as GF macrophages (about 20 erythrocytes per macrophage). In vivo-stimulated CONV macrophages also ingested particles by the Cib receptor, since endotoxin or oil treatment resulted in 70 to 80% macrophages with internalized E(IgM)C (Table 3). This did not occur in GF macrophages (Table 3). Seventy-two hours of cultivation in MEM plus 20% FCS resulted in more particles attached to both receptors in

1134 M0RLAND, SMIEVOLL, AND MIDTVEDT

IN FECT. IMMUN .

TABLE 3. Phagocytosis mediated by Fe and Cjb receptors in CONV and GF macrophages without or after in vito stimulation" GF macrophages

CONV macrophages

C( b

CGb

Fc

'e of cells

'7 of cells

Fc In vivo stimulus C

None Endotoxin Oil

of cells:

(I

of cells

Attached

Internalsized

Attached

Internalsized

Attached

Internalsized

Attached

Internalsized

93.5 ± 0.1 98.3 ± 0.1 97.0 ± 0.3

90.0 ± 0.5 96.7 ± 2.2 91.1 ± 0.8

89.3 ± 0.6 94.5 ± 2.0 92.5 ± 1.5

7.2 ± 1.3 84.1 ± 4.6 70.0 ± 3.3

96.8 ± 0.6 95.8 ± 1.4 96.1 ± 1.6

94.6 ± 0.4 95.8 ± 0.4 95.6 ± 2.4

94.2 ± 2.6 92.9 ± 1.6 93.8 ± 0.2

8.9 ± 2.1 8.3 ± 0.4 10.1 ± 0.6

"Cells were harvested 3 days after i.p. injection of 70 pg of endotoxin or 1 ml of mineral oil. Opsonized sheep erythrocytes (0.1 ml of 5%) were added to 0.5 x 10" macrophages for 30 min at 4"C for attachment and 60 min at 37"C for internalization. The results are expressed as percentage of cells that attached or internalized more than three particles (calculated by counting 500 cells per culture in the phase microscope). Means of five experiments ± standard error.

TABLE 4. Phagocytosis mediated by Fc and Cab receptors in CONV and GF macrophages after in vitro stimulation" CONV macrophages Fc

C :b

Fc

C :b

'% of cells

'7i of cells

7 of cells

'% of cells

In vitro stimulus (72 h)

Attached

FCS FCS + endotoxin "

GF macrophages

97.2 ± 0.4 97.0 ± 0.5

Internalize ized 96.8 ± 0.1 97.0 ± 0.2

Attached

Internal-

Attached

Internal-

sized

Attached

Internal-

sized

94.1 ± 1.2 96.6 ± 2.4

13.6 ± 1.9 82.0 ± 0.0

95.7 ± 1.3 96.6 ± 0.3

95.6 ± 1.3 96.0 ± 0.1

90.9 ± 3.1 98.3 ± 1.0

4.1 ± 0.9 9.5 ± 0.8

sized

Attachment and internalization were assayed as described in Table 3. Means of five experiments ± standard error.

CONV as well as GF macrophages, identical to the results for in vivo-stimulated cultures (about 20 erythrocytes per macrophage). In further agreement with the in vivo results, FCS plus endotoxin in vitro promoted complement-mediated phagocytosis in CONV but not in GF macrophages (Table 4).

DISCUSSION Previous reports demonstrated impaired DTH in germfree animals with normally functioning lymphocytes (17; 36; Lerner, Fed. Proc. 23:286, 1964). There is some evidence that changes in macrophage behavior are responsible for this impairment (14), although the studies do not indicate whether the changes are macrophage mediated only or whether disturbed lymphocyte-macrophage interactions are involved. Our findings have demonstrated that several properties of GF macrophages are different from the corresponding properties of CONV macrophages, indicating some dysfunction of GF macrophages. The difference in endotoxin-stimulated C:ib phagocytosis seems to be of special importance, since endotoxin is considered to affect macrophages directly, without any participation of T or B lymphocytes (37). It is tempting to associate the macrophage dysfunctions with the low resistance of GF animals to chronic

infections (35), as well as the lack of nonspecifical tumor cytotoxicity in macrophages from GF nude mice (19). Our data indicate, however, that resident peritoneal macrophages from GF mice were comparable to corresponding cells from CONV mice with respect to several other functions. Thus, the presence of microorganisms in CONV animals seems to have little influence on the number of macrophages in the peritoneal cavity (30), their levels of hydrolytic enzymes, or the presence of immunological receptors. These findings indicate a difference between peritoneal macrophages and other mononuclear phagocytes since Kupffer cells (5) as well as alveolar macrophages (Heise and Myrvik, Fed. Proc. 25:439, 1966) from GF rats exhibited lower amounts of lysosomal enzymes than corresponding CONV cells. In agreement with the present report, this difference was not found for peritoneal macrophages from CONV and GF rats (Heise and Myrvik, Fed. Proc. 25:439, 1966). If we assume that all types of mononuclear phagocytes are equally activated by microorganisms, the different anatomical location of the various cells may explain the difference recorded. This assumption is supported by observations of a higher state of activation of Kupffer cells and alveolar macrophages than peritoneal macrophages in CONV animals (6, 22, 31).

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MACROPHAGE FUNCTION IN GERMFREE MICE

The reason for the high level of f-glucuronidase found in the GF cells is not clear. Variations in age between peritoneal phagocytes in GF and CONV mice, caused by different influxes of young, blood-borne monocytes (14), might influence the enzyme level (2, 10). Another possible mechanism for elevated /B-glucuronidase activity in macrophages is acquisition of the enzyme from the environment (28), for instance from the intestinal bacterial flora. The mononuclear phagocyte system performs receptor-mediated endocytosis of various glycoproteins with terminal mannose or N-acetylglucosamine residues, including fB-glucuronidase (33). If bacterial enzymes compete with homologous enzyme (27) for uptake in CONV macrophages, but is degraded intracellularly (9), this might explain the higher 8l-glucuronidase assayed in GF macrophages. The only sign of low functional activity in our resident GF macrophages was reduced spreading in vitro, and fewer opsonized sheep erythrocytes attached to immunological receptors. The last function appears to be correlated with the surface area of the cells (15). Since variations in spreading have been used to characterize the functional state of macrophages (7, 8, 23), our finding may indicate at least a partial lower state of activation in resident GF macrophages than in cells from CONV mice. We found the response of macrophages to activation, both in vivo and in vitro, to be markedly modified by the germfree status of the mouse. The reduced chemotactic response obtained after i.p. oil injections in our study was supported by previous findings of Jungi and McGregor (14). In this and previous studies (7, 23), phagocytosis of C3b-opsonized erythrocytes could be induced in CONV peritoneal macrophages by stimulating the cells in vivo with E. coli endotoxin or oil, or in vitro with endotoxin in the presence of FCS. No similar induction was obtained in macrophages from GF mice. The role of C3b-mediated ingestion in chronic infections and DTH is not well understood. It makes clearance of antigenic particles more efficient, since both F and C3b receptors are able to ingest the particles. This may be important during infections, where IgG may absorb to the Fc receptors and thus impair the ingestion of IgG-coated particles, such as bacteria, in macrophages. Many microorganisms produce or possess materials which activate complement; therefore they may become coated with C3b and ingestible for activated macrophages (J. A. Griffin and F. M. Griffin, Jr., personal communication). If the C3b-mediated phagocytosis is indeed linked to the ability of activated macrophages to handle bacteria, then the response to bacterial endo-

1135

toxin may be considered as some kind of secondary reaction in the macrophages. It is, however, pertinent to point out that GF macrophages, collected after i.p. injections of oil, still failed to internalize particles via C3b receptor. This indicates a more general failure of GF macrophages to respond to foreign stimuli. It must, however, be taken into account that GF and CONV mice might possess different peritoneal cell populations after activation in vivo, which could explain the difference in C3b phagocytosis. Ruco and Meltzer found about 10-foldhigher response to lymphokines in young peroxidase-positive mononuclear phagocytes, newly recruited from the circulation, than in resident cells (26). However, we also obtained activation of the C3b receptor in vitro on CONV cells, which indicates that the resident CONV tissue macrophages can be induced to ingest complement-coated particles, whereas this capacity is not found in GF macrophages. Bianco et al. previously proposed covariation of C3b-mediated internalization and macrophage activation (7). Our report on macrophage activation, including studies of lysosomal enzyme activities, lead to the same conclusion (23). The present findings do not fit completely with this proposal, since increased intracellular activity, as well as selective release (29) of lysosomal enzymes, could be induced by appropriate activating agents in GF macrophages, whereas C3bmediated phagocytosis could not. Quantitatively, the increase in enzyme activities was indeed lower in GF than in CONV macrophages, but prior contact with microorganisms is obviously not essential for induction or secretion of lysosomal enzymes. ACKNOWLEDGMENTS We thank Annelise Gr0holt, Ingrid Fagerheim, Anja Pedersen, and Randi Ystborg for excellent technical aid, and Annikken Gulseth for skillful help in preparing the manu-

script. LITERATURE CITED 1. Abernathy, R. S., and J. J. Landy. 1967. Increased resistance to endotoxin in germ-free guinea pigs. Proc. Soc. Exp. Biol. Med. 124:1279-1283. 2. Bainton, D. F., B. A. Nichols, and M. G. Farquhar. 1976. Primary lysosomes of blood leucocytes, p. 3-32. In J. T. Dingle and R. Dean (ed.), Lysosomes in biology and pathology, vol. 5. Elsevier-North Holland Publishing Co., New York. 3. Barrett, A. J. 1972. Lysosomes, p. 111. In J. T. Dingle (ed.), A laboratory handbook. North-Holland Publishing Co., Amsterdam and London. 4. Bauer, H., F. Paronetto, W. A. Burns, and A. Einheber. 1966. The enhancing effect of the microbial flora on macrophage function and the immune response. A study in germfree mice. J. Exp. Med. 123:1013-1026. 5. Berg, T., and T. Midtvedt. 1976. The influence of infection on the content of lysosomal enzymes in rat Kupffer cells and hepatocytes. Acta Pathol. Microbiol. Scand. Sect. A 84:415-420.

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Comparison of peritoneal macrophages from germfree and conventional mice.

INFECTION AND IMMUNITY, Dec. 1979, 0019-9567/79/12-1 129/08$02.00/0 p. 1129-1136 Vol. 26, No. 3 Comparison of Peritoneal Macrophages from Germfree...
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