Macrophage activation during experimental murine brucellosis

CELLULAR

IMMUNOLOGY

Macrophage

49, 154- 167 (1980)

Activation

during

Experimental

Murine

II. Inhibition of in Vitro Lymphocyte Proliferation Brucella-Activated Macrophages CLARE Department

of Microbiology,

RIGLAR~ University Received

AND CHRISTINA of Melbourne, February

Brucellosis by

CHEERS?

Parkville,

Victoria,

3052, Australia

9, 1979

During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.

INTRODUCTION Brucellae are facultative intracellular bacteria which cause chronic infections in a wide range of mammalian species. The organisms survive within the phagocytic cells of the reticuloendothelial system, and, as with mycobacteria, salmonellae, and Listeria (l), the elevation of antibacterial activities of the macrophages is an important feature of the immune response (2,3). Mice have provided useful, easily manipulated models for these intracellular infections, although they are not necessarily the natural host. In experimental murine tuberculosis and listeriosis, it has been shown that macrophage activation is a T-cell-dependent function (4, 5), that macrophages proliferate at the site of infection (6,7), and that monocytes are recruited to the site from the bone marrow (4, 8). It seems from the present experiments that macrophages also accumulate at the site of Brucella infection of mice. A feature of all these infections, with the possible exception of listeriosis, is their chronicity. Despite the persistence of relatively high numbers of organisms, macrophage activity in murine brucellosis declines after an early peak of efficiency ’ Present address: Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia. 2 To whom all correspondence should be addressed. 154 0008-8749/80/010154-14$02.00/O Copyright All rights

0 1980 by Academic Press, of reproduction in any form

Inc. reserved.

SUPPRESSOR

MACROPHAGES

DURING

BRUCELLOSIS

155

(9, 10). It is therefore of interest to study possible feedback control mechanisms which may limit the duration or effectiveness of cell-mediated immunity. One such possible feedback control is mediated by macrophages themselves. In a variety of situations where macrophage numbers are elevated above normal, and particularly where macrophages are “activated,” inhibition of various in vitro lymphocyte responses has been observed (reviewed by Nelson (11)). A number of examples have been noted in which injection of the donor animals with live (12- 15) or killed (16, 17) bacteria, with viruses (18) or with parasites (19, 20), causes accumulation of activated macrophages in the spleen or peritoneal cavity, variously suppressing in vitro antibody production, mitogen responsiveness, mixed lymphocyte reaction, generation of cytotoxic T cells, or the ability to transfer GVH. The present study describes apparently similar findings in CBA mice infected with Brucella abortus strain 19. A succeeding paper considers whether this phenomenon is relevant to the in vivo course of infection (21). MATERIALS

AND METHODS

Mice

CBA/H mice originally from the Walter & Eliza Hall Institute, Melbourne, Australia were maintained by pedigreed brother-sister mating in the Microbiology Animal Breeding Unit. In each experiment mice of the same sex were infected at 5-7 weeks of age. Bacteria Brucella abortus (strain 19), a smooth attenuated strain, was obtained from Commonwealth Serum Laboratories, Melbourne. Cultures were maintained by weekly subcultures on horse blood agar (HBA)3 and renewed after fewer than 50 transfers. Znfection of Mice

Bacteria were washed from 24-hr nutrient agar cultures with 1% horse serum in distilled water (serum water). The suspension was standardized turbidometrically to give approximately 5 x lo5 brucella in 0.2 ml and injected intravenously. The dose was confirmed by a viable count (22). Enumeration of Organisms in the Spleen

Spleens were homogenized in an MSE microhomogenizer and aliquots of lo-fold serial dilutions in serum water were plated on nutrient agar or HBA. Colonies were counted after 3 days incubation at 37°C. Tissue Culture Media

Eagles minimum essential medium (Grand Island Biological Company, Cat. No. F-15) was made up with 9.6 g/liter powdered medium, 60 mg/liter penicillin, 100 3 Abbreviations used: Con A, concanavalin A; FCS, fetal calf serum; 3Haa, tritiated amino acid mixture; HBA, horse blood agar; HMEM, Hepes-buffered Eagle’s minimum essential medium; [3H]TdR, tritiated thymidine; LPS, lipopolysaccharide; MEM, Eagle’s minimum essential medium; PHA, phytohemagglutinin; serum water, 1% horse serum in distilled water; TdR, thymidine.

156

RIGLAR

AND CHEERS

20 TIME

40 SINCE

60 INFECTION

(days)

FIG. 1. Growth of &UC& and splenomegaly in CBA mice infected intravenously with 5 x 10SB. strain 19. (0 - - - 0) infected mice. (0 0) normal controls. Bacterial numbers are expressed as geometric means, spleen weights as arithmetic means. Vertical bars represent the standard deviation of five mice. abortus

mg/liter streptomycin, 5 x lop5 M 2-mercaptoethanol, and 10% fetal calf serum (FCS). The medium was buffered with bicarbonate, 3.56 g/liter (MEM) or for nylon wool filtration of cells, with Hepes, 0.02 M, pH 7.2 (HMEM). For HMEM, mouse cell osmolarity was maintained by dissolving 9.6 g powder in 800 ml. Brucella

Antigen

Brucellin was prepared from B. abortus strain 19 by the method of Bhonghibhat et al. (23). Protein was assayed by the Lowry method (24). The 150 mg was nonpyrogenic in the USP rabbit test, although 13 pg of Escherichia coli 011 l:B4 lipopolysaccharide (Difco) was, suggesting that there was less than 90 pg endotoxin/mg protein. On the other hand, the limulus amoebocyte lysate test (Microbiological Associates, Md.) indicated more than 8 pg endototoxin/mg (25). Cell Culture Spleen cells were teased gently through go-mesh wire sieves into cold MEM. Debris was allowed to settle through a cushion of FCS, and erythrocytes

FIG. 2. Hematoxylin-eosin stain of spleen of mouse strain 19, showing accumulations of large mononuclear Magnification (top 400x, bottom 100x).

infected 21 days earlier with 5 x cells at the junction of the white

1o”B. abortus and red pulp.

158

RIGLAR

AND CHEERS

20

II d 0

$00

750

1000

1250 CELL

FIG. 3. Size distribution of cells from uninfected spleen (-) days earlier with 5 x lo5 B. abortus strain 19 (- - -).

1500 VOLUME

1750 (p3)

and from spleen of mouse infected 14

were lysed with Tris-buffered 0.83% ammonium chloride, pH 7.2 (26). The cells were centrifuged through an FCS cushion and resuspended in MEM, and viable nucleated cell numbers were determined by eosin exclusion. Peritoneal cells were washed from the unstimulated peritoneal cavity with 2 2.5ml vol of MEM containing 1 part per 100 heparin (preservative free, Commonwealth Serum Laboratories, Melbourne, Australia). The cells were spun through an FCS cushion and resuspended in MEM without heparin. Microcultures containing 2 x lo5 or 5 x lo5 viable spleen cells in 0.2 ml were set up in microtiter trays with flat-bottom wells (Falcon Plastics). Mitogens were generally used in the following concentrations per well: brucellin, 5 pg; phytohemagglutinin (PHA) (Wellcome), 0.3 Fg; PHA (Difco), 0.05 ~1; concanavalin A (Con A) (Miles Laboratories Inc.), 1 pg; lipopolysaccharide (LPS) from Escherichia coli 011 l:B4 (Difco), 2 pg. Cultures were incubated at 37°C in a humidified atmosphere with 10% CO, for 3 days. Tritiated thymidine ([3H]TdR) was added 18 hr before harvesting. In most experiments 1 FCi [3H]TdR of specific activity 5 Ci/mM (Amersham) was used per well. In some experiments (indicated under Results) 0.025 &i of 26 Ci/mM [3H]TdR (C.E.A. France, Service des Molecules Marquees) was used. In one experiment a tritiated amino acid mixture (Amersham Catalog No. TRK 440) was used. For some experiments double-chambered culture vessels (27) were prepared with an inner and outer chamber separated by either dialysis tubing (Union Carbide Corporation, Chicago, Ill.) or a Nuclepore membrane of 0.02-/*rn pore size (General Electric Co., Pleasanton, Calif.). The outer chamber was separated from 50 ml of MEM by dialysis tubing. The level of fluid in all chambers was equal. Cells were added to the inner chamber at a concentration of 1 x lo6 in 1 ml, and 2.2 x lo6 cells in 2 ml were added to the outer chamber. Brucella antigen (200 wg) and 5 $X3H]TdR of 5 Ci/mM were added to the two compartments according to the volume ratio. Microtitre tray cultures were harvested onto filter disks (Whatman, GF/C) with suction in a multiple-sample harvester (28) and wells rinsed with isotonic saline. Double-chamber cultures were harvested manually onto filters. The filtered cells

SUPPRESSOR

MACROPHAGES

10

20

DURING

30

TIME

159

BRUCELLOSIS

40

SINCE

50

INFECTION

(days)

60

FIG. 4. In vitro proliferation of spleen cells from 19. abortus-strain-l9-infected mice, with 5 x IO” cells/culture, 3-day culture period and 0.025 &i L3H]TdR added. Cultures were unstimulated (0-O) or stimulated by addition of 5 pg brucellin (0 - - - 0). Vertical bars represent standard deviation of six cultures.

were washed with distilled water, spreading the DNA onto the filter paper and removing excess label. The papers were dried and added to 10 ml toluene containing 5 g/liter PPO and 0.1 g/liter POPOP in glass vials and counted in a Packard Tri-Carb liquid scintillation spectrometer, Model 3320. Size Range of Spleen Cells Scans of spleen-cell size were performed on a Model B Coulter cell counter. Data obtained as relative cell number in discrete size intervals was normalized by plotting the percentage of total cell number for each size interval, after exclusion of the low size intervals in which cell debris and nonnucleated cells appeared. Adherent

Cell Removal

(a) Nylon wool columns. This method was adapted from that described by Julius, et al. (29). Nylon wool columns were prepared with 0.3 g nylon wool (LP-1 Leukopak Leukocyte Filter, Fenwal Laboratories, Ill.) packed to the 2-ml mark of a 5-ml plastic syringe, which had a 25-guage needle connected to the outlet by a short length of surgical tubing closed with a clamp. The whole assembly was autoclaved before use, then rinsed with HMEM and allowed to equilibrate to 37°C. The columns were rinsed with 6 ml of warm medium, then 5 x lo7 cells in 0.2 ml of HMEM were added to the column and washed in with 1 ml of warm HMEM. After stationary incubation for 5 min the cells were washed through the column at 1 ml/min and 10 ml of eluate was collected. The cells were then spun down and resuspended in MEM for culture. (6) Settling on plastic. Adherent cells were removed from cell suspensions by repeatedly settling 1 x 10’ cells in plastic dishes (35 mm diameter, Falcon Plastics) for 30 min at 37°C and then removing the nonadherent cells. A sample of nonadherent cells was cultured after each step.

160

RIGLAR

AND CHEERS TABLE

Proliferation”

1

of Spleen Cells from Normal and 20-Day Bruce/la-1PInfected in Response to Various Mitogens

Mice

Cells Mitogen”

Normal

None

18,870 95,680 90,090 219,608 149,308

antigen Phytohemagglutinin Concanavalin A Lipopolysaccharide Bruce/la

(1.17) (1.14) (1.04) (1.13) (1.14)

20 day infected 6,383 13,130 11,700 106,400 56,920

(1.15) (1.37) (1.26) (1.37) (1.21)

n Proliferation measured in 3-day cultures of 2 x lo5 cells with 1 &i [3H]TdR (5 Ci/mM). b Brucella antigen 5 &culture, PHA (Wellcome) 1.5 pgkulture, Con A 1 &culture, and LPS 2 &culture. c Geometric means of four cultures. Multiplication factor to derive upper and lower limits of standard deviation shown in parentheses.

Determination

of Phagocytic

Cell Number

The number of phagocytic cells present in suspensions was estimated by uptake of fluorescein-labeled heat-aggregated bovine serum albumin (30). RESULTS Course of Brucellosis

in the Spleen

CBA mice were infected at 5 weeks of age with 5 x lo5 B. abortus strain 19. Figure 1 compares the growth of bacteria in the spleen with splenomegaly over a period of 60 days. Brucella 19 grew to peak levels by Day 7 of infection then showed little change in numbers until Day 14. Between Days 14 and 20 numbers were reduced more than lo-fold but then there was little change until after the 42nd day when the infection began to decline. Marked splenomegaly was observed, with the spleen weight rising to a peak of 0.49 g (5.93 times normal) at 21 days postinfection. The weight of spleens in matched, uninfected mice did not vary during this period. Associated with the increased spleen weight, there was altered histological appearance with increased numbers of large, pale-staining cells observed at the periphery of the lymph follicles in hematoxylin- and eosin-stained sections (Fig. 2). In addition the cell-size distribution in the spleen 14 days postinfection was altered, with an increase in numbers of relatively large cells as measured by a Coulter B-cell size scan (Fig. 3). The proportion of phagocytic cells in the spleen of 21-day infected mice was estimated to be 7% compared with less than 1% in normal mice. In Vitro Lymphocyte

Proliferation

The effect of Brucella infection on proliferation of spleen cells in vitro in response to brucellin was examined. Proliferation was measured by incorporation of [3H]TdR in cultures of 5 x lo5 whole spleen cells taken from mice at various stages of infection. In cultures of cells from mice infected with Brucella 19 for 2 to 3 weeks, proliferation with or without added brucellin was greatly reduced compared with

SUPPRESSOR

MACROPHAGES

DURING

~105

5x105

1 x106

161

BRUCELLOSIS

I

I

5

50250

5

50 250

5

50 250

,

I

I

2

1

I

5

I

3

2

MITOGEN

/WELL

(pg)

FIG. 5. In vitro proliferation of 1 x 106, 5 x 105, or 2 X loj spleen cells from normal (-) 20-day-infected (- - -) mice in the presence of graded doses of mitogen. Top: brucellin. Center: Bottom: Con A. Background counts without mitogen have been subtracted.

or LPS.

that of normal cells (Fig. 4). Little or no stimulation in the presence of brucellin was observed during the period of maximum inhibition. Reduced proliferation also occurred in response to nonspecific mitogens in cultures of spleen cells from mice infected 20 days earlier with brucella 19 (Table 1). To see whether this suppression occurred under all conditions of culture, varying numbers of cells from normal or 20-day infected mice were cultured with different concentrations of mitogens. In Fig. 5, for clarity of presentation, background counts in unstimulated cultures have been subtracted from those in cultures with

162

RIGLAR

AND

CHEERS

mitogen. Particularly with LPS and Con A, suppression was greater in cultures with higher numbers of cells. Furthermore, suppression could be overcome by adding more mitogen. Excessive amounts (12 pg) of Con A were apparently toxic for both normal and infected spleen cells, reducing uptake of [3H]TdR to below-background levels. Cell Mixing

Experiments

Cells from mice infected 20 days earlier were able to suppress the response of normal spleen cells when equal numbers of cells from normal and 20-day infected mice were cultured together (Table 2). However, cells from mice infected 26 days earlier, although still showing some reduction in their own responsiveness, were unable to suppress significantly the response of normal cells. The 26-day spleen cells were not refractory to suppression by cells from 20-day infected spleen. Removal

of Adherent

Cells

The large number of macrophage-like cells visible in spleen sections suggested a role for adherent cells in inhibition of proliferation. The effect on spleen-cell proliferation of removing adherent cells by nylon wool filtration and by settling onto plastic was examined. Nylon wool filtration (Table 3A) had little effect on proliferation of normal cells but caused a marked increase in proliferation of cells from 20-day-infected mice cultured withBrucella antigen and restored proliferation levels to those of normal cells in the case of cultures with the nonspecific mitogens PHA and LPS. Removal of adherent cells from normal spleen by settling onto plastic generally caused a slight decrease in proliferation by normal cells. However, when adherent cells were removed from infected spleen populations by this method a distinct increase in proliferation was observed after three or four repeated settling steps (Table 3B). TABLE Suppression

of Normal Infected

2

Spleen-Cell Proliferation by Spleen Cells 20 or 26 Days Earlier with Brucella 19” Unstimulated

Cells Normal 20 day 26 day NormaP + 20 day Normal + 26 day 20 day + 26 day

cpm* 932 123 398 200 511 190

(1.22) (1.19) (1.17) (1.27) (1.09) (1.21)

Brucella

Suppression 0.38 0.77 0.73

index”

cpmb 2571 142 908 321 1457 254

(1.03) (1.21) (1.17) (1.23) (1.15) (1.42)

from

Mice

antigen

5 &culture

Suppression

indexC

0.24 0.84 0.48

n Proliferation measured in 3-day cultures of 5 x lo5 cells with 0.025 /Xi VH]TdR (26 Ci/mM). b Geometric mean of four cultures. Multiplication factor to derive the upper and lower limits of the standard error shown in parentheses. c Ratio observed/expected cpm, i.e., cpm population a + b/M(cpm population a + cpm population b). d Equal numbers of the two populations were cocultured.

SUPPRESSOR MACROPHAGES TABLE Proliferation”

Cell source

Nil Filtered Nil Filtered

B. Normal Normal Infected Infected Infected

Nil Settled X3 Nil Settled X3 Settled X4

163

BRUCELLOSIS

3

in Cultures of Spleen Cells from Normal and Bruce/la-19-Infected Mice after Removal of Adherent Cells

Method of removal of adherent cells

A. Normal Normal Infected Infected

DURING

Mitoge# Brucella

None

antigen

2,642 (1.12)e 4,319 (1.10) 1,825 (1.13) 4,032 (1.13) 101 (1.16) 58 (1.13) 650 (1.28) 2,223 (1.12) 27,520 18,250 8,030 8,183 42,580

PHA 3,274 3,357 327 3,224

Con A

(1.11) (1.08) (2.38) (1.10)

(1.11) (1.16) (1.06) (1.02) (1.07)

469,100 353,100 113,300 311,700 314,200

(1.39) (1.09) (1.24) (1.10) (1.08)

LPS 3,772 3,447 537 3,279

(1.11) (1.17) (2.49) ( 1.05)

248,600 271,000 128,800 164,300 208,200

(1.08) (1.43) (1.03) (1.01) (1.12)

u Proliferation measured in 3-day cultures of 5 x lo5 cells with 0.025 yCi [3H]TdR (26 Ci/mM) (experiment A) or of 2 x lo5 cells with 1 &i [3H]TdR (5 Ci/mM) (experiment B). * Brucel/a antigen 5 pg/culture, PHA (Difco) 0.5 phculture, Con A 1 CLg/culture, LPS 2 &culture. ’ Geometric means of six cultures. Multiplication factor to derive upper and lower limits of standard deviation shown in parentheses.

Addition of Adherent Cells

An alternative approach to determine the role of adherent cells in inhibition of proliferation was to add adherent cells to normal cell cultures. Graded numbers of spleen cells from 21-day-infected mice were allowed to settle overnight in the culture wells and nonadherent cells were removed by gentle but thorough washing. To the remaining adherent cells were added normal spleen cells. Reduced proliferation was observed (Fig. 6) with maximum reduction when adherent cells from a suspension of 4 x lo5 viable spleen cells were added.

6“\i

4, 0 12

4 CELLS

x) SETTLEDdO-5

FIG. 6. In vitro proliferation of 2 x lo5 normal spleen cells in the presence of adherent splenic cells from infected mice. Graded numbers of spleen cells from infected mice were allowed to settle in culture overnight, nonadherent cells removed, and normal spleen cells added together with mitogen. (O-O) unstimulated; (0 0) brucellin; (A A) LPS; (Cl 0) Con A. Each point represents the geometric mean of uptake of 1 &i [3H]TdR in four cultures plus or minus standard deviation.

164

RIGLAR AND CHEERS

Effect of Cell Separation

on Mediation

of Inhibition

In order to investigate the presence of a soluble inhibitory factor, spleen cells from normal or infected mice were cultured in a double-chambered system in which the cells were separated by either dialysis or Nuclepore membranes (Table 4). Inhibition of proliferation of normal cells in the inner chamber by infected cells in the outer occured to the same extent across either of the two membranes (inhibition index = 52 or 54%). The inhibition was somewhat stronger when the cells were mixed directly in the same proportions (inhibition index = 67%). In turn, inhibition with the cell mixture was less than that with cells from infected mice alone (see counts for outer chambers). Is the Inhibitor Cold Thymidine? A trivial explanation of the observed inhibition of thymidine uptake is that macrophages release cold thymidine which competes with the radiolabeled thymidine (31). Therefore, cultures were assayed in the presence of different amounts of labeled or cold thymidine. Table 5 shows that the inhibition of proliferation amongst cells from 20-day-infected mice is the same over a 500-fold range of thymidine doses. In contrast, when 1 x lo5 peritoneal cells were added to 5 x lo5 normal spleen cells, inhibition of proliferation was almost absolute when 1 or 5 &i [3H]TdR (0.2 or 1 mmol thymidine) was added, but when 100 mmol cold thymidine was included inhibition was greatly decreased. Inhibition of incorporation of amino acids was demonstrated in both systems. DISCUSSION Infection of mice withB. abortus strain 19, which survives mainly in the spleen, activates the bactericidal mechanisms of splenic macrophages (9) and leads to the accumulation of large numbers of macrophage-like cells in the spleen (Figs. 2 and 3). Macrophage activation peaks 14 days after infection (9, 10) and results in the removal of more than 90% of the infecting organisms. However, about lo6 bacteria per spleen remain. Despite this antigenic load, macrophage activation, a T-lymphocyte mediated phenomenon (Cheers, unpublished), declines (9, 10). TABLE Proliferation”

4

in Cultures Separated by a Cell-Impermeable cpm [‘H]TdR Inner

Membrane incorporated

chamber

Outer

chamber

Cell type Membrane Inner

Outer

NOlllld + infected NOllttd NOlllld NOllttd Normal

NOlld + infected Infected Infected NORtX4l NOlltld

type

Dialysis Dialysis NUdepore Dialysis NUCkpOR

Unstimulated

24,870 (1.55)” 86,640 (1.51) 68,140(1.29) 178,000 (1.26) 166,7cnl(1.51)

Bruce/la antigenC

63,100 (1.13) 175,100 (1.05) 166,500 (1.50) 369,300 (1.20) 372,700 (1.58)

Unstimulated

29,490 20,060 27,460 198,200 261.600

(1.22) (1.33) (1.67) (1.35) (1.23)

Bruce/la antigenP

134,700 41,120 85,060 482.600 458,100

(1.20) (1.42) (I .59) (1.27) (1.10)

n Proliferation measured in 3-day cttlh~res of 10 x IV cells/inner chamber, 22 x 10s cellsiouter chamber with 5 &i PHITdR (5 CilmM). b Cells from normal mice or from mice infected 20 days earlier with BruceNo 19. c The 2CNl m Brucefla antigen added to inner chamber, 425 pg to outer. * Geometric mean of three cultures. Multiplication factor to derive upper and lower limits of standard deviation shown in parentheses.

SUPPRESSOR MACROPHAGES

DURING

TABLE

165

BRUCELLOSIS

5

Effect of Varying Concentrations of Thymidine on Observed Suppression of Proliferation 100 &f TdRb (5 @.Zi [‘HlTdR)

CellS” Normal spleen Infected spleen Normal spleen + peritoneal cells

cpm

I pM TdR (5 &i [3HlTdR)

Inhibition’ (96)

1,521 (1.37)” 559 (1.20)

-

701 (1.47)

54

64

0.2 LLM TdR (I &i 13HlTdR)

Inhibition’ W)

i pCi ‘Haa

CPm

InbibitlOnC (%)

11,414(1.04) 5,515 (1.59)

52

3,726 (1.45) 1,458 (1.23)

61

1.206(1.14) 5% (1.35)

54

719 (1.24)

94

222 (1.31)

94

571 (1.34)

57

cw

cm

Inhibitionc 6%)

n A total of 5 x It? cells/well were cultured with 5 pg brucellin. The cells were from the spleens of normal or 20.day Brurelluinfected mice. Normal spleen cells and normal peritoneal cells were mixed in a ratio of 3: I. * At 18 hr before harvesting. c3H]TdR (5 Ci/mM) or Wlabeled amino acids (JHaa) were added. Excess cold thymidine was added to some cultures to vary the final concentration of thymidine (TdR). r Percentage inhibition in cultures of spleen cells from infected nuce or of cultures containing an excess of peritoneal cells compared with normal proliferation. * Geometric mean of radioactive uptake in triplicate cultures. Multiplication factor to derive upper and lower limits of standard deviation shown in parentheses.

There is evidence that its recall by challenge with further B. abortus is suppressed (10, 32). It was the search for possible suppressor mechanisms which lead us to examine the role of macrophages themselves in feedback inhibition of lymphocyte responses. The extensive literature on macrophage suppression of lymphocytes has been reviewed by Nelson (11). It includes a number of examples where infection has lead to the accumulation of large numbers of activated macrophages which can suppress various in vitro parameters of lymphocyte responsiveness (12-21). The present experiments indicate that, duringBrucella infection of CBA mice, the accumulation of macrophage-like cells in the spleen caused gross splenomegaly (Fig. 1). In parallel with this, the response of cultured spleen cells to brucellin was suppressed (Fig. 4). It is unclear how brucellin stimulates lymphocyte proliferation. It probably acts as a nonspecific B-cell mitogen, since uptake of [3H]TdR in response to brucellin was of the same order as for other mitogens, and since proliferation of spleen cells from thymusless “nude” mice can be stimulated by the brucellin (Tuan Mu and Cheers, unpublished). The brucellin preparation used contained between 8 and 90 pg endotoxin/mg protein measured by pyrogenic reaction in rabbits and limulus amoebocyte lysate test (25). While 0.45 pg LPS per culture is enough to stimulate cell proliferation, Brucella endotoxin has been shown not to stimulate in vitro proliferation of lymphocytes (33). Responsiveness to other mitogens was also suppressed (Table 1). Inhibition of proliferation could be overcome by increasing the amount of mitogen added per culture (Fig. 5), indicating that the cells remained viable. Background cell proliferation was also decreased (Fig. 4 and Table l), suggesting a general suppression of cell proliferation. Cell mixing experiments showed that the spleen-cell population from infected mice included a component which was suppressive to normal spleen cells (Table 2). This was apparently an adherent cell, since removal of nylon wool- or plasticadherent cells restored the response to normal (Table 3). Since in vitro proliferation

166

RIGLAR

AND

CHEERS

in response to mitogens generally requires macrophages (34), it can only be assumed that we were observing a relative removal of adherent cells, rather than an absolute. There was no loss of adherent B lymphocytes (29), as indicated by the constant response to LPS. These findings were supported by experiments in which adherent cells from the spleens of 20-day infected mice were added to cultures of normal spleens and shown to inhibit proliferation (Fig. 6). Maximal inhibition was achieved when the adherent cells from 4 x lo5 viable spleen cells were added to 2 x lo5 normal cells. It was estimated that 7% of the cells from infected spleens were phagocytic. Many suggestions have been put forward for the possible mechanism whereby macrophages inhibit the response of lymphocytes in vitro, including excretion of prostaglandin (35), interferon (36, 37), arginase (38), or other uncharacterized soluble factors (11). A criticism of experiments in which lymphocyte responsiveness is measured by uptake of tritiated thymidine is that macrophages lack thymidine kinase (39) and thus secrete unlabeled thymidine (3 1). This may compete with tritiated thymidine and produce the illusion of inhibition. However, in the present system it was shown that inhibition could be demonstrated equally well in the presence of 500-fold excess thymidine to overcome possible secretion of thymidine or measured by the uptake of tritiated amino acids (Table 5). This was in contrast to the situation where large numbers of normal peritoneal macrophages were added to the culture in which case inhibition was greater with less thymidine. Even here some degree of true inhibition occurred as measured by tritiated amino acids or high concentrations of thymidine. We wished to investigate the presence of an inhibitory material in the supernatant of cultures of spleen cells from infected mice. Unfortunately, in common with others (11,20), we found that supematants from the cultures of normal spleens were equally inhibitory. Therefore, we prepared cultures in which normal spleen cells were separated from cells of 20-day-infected mice by dialysis or Nuclepore membranes (Table 4). The pore size of the membrane made no difference to the inhibitory effect. Since brucellin is not dialyzable it seems that macrophages are not acting by removing mitogen, a possibility which might have explained the observed effect of adding more mitogen (Fig. 5). Nevertheless, inhibition was somewhat more efficient when the cells were mixed in the one chamber (inhibition index = 69%) than when separated by membranes (inhibition index = 52, 54%). This may indicate a relatively labile, short-range factor. Hence we have shown that Brucella infection of mice causes the accumulation in the spleen of macrophage-like cells which, as well as mediating bactericidal activity against the pathogen, can inhibit the in vitro responsiveness of lymphocytes to a number of mitogens, including one derived from Brucefla organisms themselves. Inhibition is apparently mediated by a low-molecular-weight, possibly labile factor. Whether this inhibition plays any role in the production of chronic infection is discussed in the following paper (21). ACKNOWLEDGMENTS This work was supported by the National Health and Medical Research Council of Australia and the University of Melbourne Restricted Purposes Medical Research Funds. We wish to thank Dr. Ken Shortman of the Walter and Eliza Hall Institute for performing the cell scans on the Couher counter and Mr. R. Cartwright for performing rabbit pyrogen tests.

SUPPRESSOR MACROPHAGES

DURING BRUCELLOSIS

167

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