CD4+ T cells mediate preexposure-induced increases in murine intraabdominal abscess formation

CLINICAL

Vol.

IMMUNOLOGY

77, No.

1, October,

AND

IMMUNOPATHOLOGY

pp. 82-88,

1995

CD4+ T Cells Mediate Preexposure-Induced Increases in Murine lntraabdominal Abscess Formation’ ROBERT G. SAWYER,* *Department

of Surgery,

REID

B. ADAMS,?

ADDISON

of Michigan of Virginia

Medical Health

University University

K. MAY,? School, Sciences

Relatively minor infectious insults are common events in today’s increasingly ill patient population. Although immunological changes are well documented during and after severe illness or stress (l-41, the effects of less serious, non-life-threatening infections on the immune system and subsequent disease have not been investigated fully. To study this issue, we have developed a model of transient bacterial infection followed by a more serious and chronic infection, previously reporting that mice exposed to live Escherichia coli or Bacteroides fragilis develop a greater number of intraperitoneal abscesses

and 22908

TIMOTHY TDepartment

I,.

PRUETTt of Surgery,

METHODS

Animals,

Bacteria,

and Fecal Adjuvant

Male Balb/c mice weighing 20-25 grams (Charles River Laboratories, Wilmington, MA) were used for all experiments. Animals were housed in stainless steel wire cages and received lab chow and water ad lib. according to National Research Council standards. All experiments were approved by the University of Virginia Animal Use Committee. E. coli 0065 (018, K-, H-nonmotile), a clinical isolate, was grown from a single colony off a stock plate by

of of

82 Inc. reserved.

48109-0331; Virginia

AND

after a subsequent mixed E. coli-B. fragilis peritonitis (5). This preexposure, performed 2 and 1 week prior to the induction of peritonitis, is well tolerated, produces increased numbers of abscesses if given intraperitoneally, systemically, or subcutaneously, and leads to no change in eventual overall mortality (5,6). Further experiments have demonstrated that this response is neither antigen specific nor reproducible with lipopolysaccharide alone, yet appears to be limited to certain gram-negative bacteria (7). Insight into the immunology of abscess production has been gained over the past 16 years in a model of B. fiagilis intraabdominal abscesses. These abscesses are completely preventible by immunization with B. fragiZis capsular polysaccharide complex (8). Through a series of passive transfer experiments, this immunity appears to be mediated by both CD4’8’ and CD8’ lymphocytes and a 14-kDa soluble protein (9-16). The polysaccharide complex responsible for abscess formation has also been further isolated and characterized (17). These experiments implied the cellular immune system could be critical to the effect on abscess formation we have seen in our system. The studies described in this paper were designed to examine changes in lymphocyte population produced after simple bacterial preexposure and deduce, via passive transfer experiments, alterations in the host immune system responsible for the eventual effects seen on later peritonitis and abscess formation.

INTRODUCTION

0090-1229/95 $12.00 Copyright 0 1995 by Academic Press, All rights of reproduction in any form

K. ROSENLOF,t

Ann Arbor, Michigan Center, Charlottesville,

We have previously shown that an increased number of Escherichia coli/Bacteroides fragilis intraabdominal abscesses are produced in mice after preexposure to small numbers of live E. coli or B. fragilis. Splenic lymphocyte subset changes and the importance of different elements of the immune response in this system were studied. Preexposure to bacteria induced a significant increase in the percentage of splenic T cells without altering the CD4/CD8 ratio. The passive transfer of either 10’ mixed splenic lymphocytes, 5 x lo6 mixed T cells, or 2.5 x 10’ CD4’ T cells from preexposed animals to naive siblings 24 hr prior to abscess induction resulted in increased abscess formation. Transfer of serum, B cells,
* This work was supported by an Infectious Diseases Society America/Stuart Pharmaceutical Fellowship grant and University Virginia Research and Development Grant 5-507-RR05431-28.

LYNN

CD4’

LYMPHOCYTES

AND

incubating in brain-heart infusion (BHI) broth (Becton Dickenson and Co., Cockeysville, MD) for 18 hr in a 37°C oscillating water bath. Cultures were centrifuged for 12 min at 2400g at 4”C, and pellets were washed twice with 0.9% saline. Bacteria were quantified using serial dilutions and plate enumerations. B. frczgilis (ATCC No. 23745) was prepared by inoculating 1 ml of a stock chopped meat broth solution into BHI broth supplemented with hemin and menadione (CarrScarborough, Stone Mountain, GA) and incubated anaerobically for 18 hr at 37°C. Cultures were centrifuged for 12 min at 2400g at 4”C, and pellets were washed twice in prereduced Hanks’ balanced salt solution (Sigma Chemicals, St. Louis, MO). Bacteria were quantified by plate dilution on brucella agar (Becton Dickenson) in an anaerobe chamber. Sterile fecal adjuvant solution was made by grinding dried mouse feces into a fine powder, adding to 0.9% saline to form a 5% (wtivol) mixture, and sterilizing in a steam autoclave at 121°C for 20 min. This preparation was refrigerated at 4°C and sterility was assured prior to each use by negative culture on blood agar plates. Immunization Procedure Splenic Lymphocytes

and Harvesting

of

Mice were inoculated with lo3 live E. coli, lo5 live B. or a combination of the two, via intraperitoneal injections (total volume, 100 ~1) 2 and 1 weeks prior to harvesting of splenic lymphocytes. Neither control animals nor animals to be sacrificed as a source of naive lymphocytes or serum received pretreatment. Previous experiments have demonstrated that intraperitoneal injections of vehicle (saline or prereduced Hanks’ balanced salt solution) do not affect later intraabdominal abscess formation. There were no deaths from these inoculations, examination of killed animals revealed no discernible intraabdominal pathology, and cultures of the peritoneum, liver, and spleen 1 week after such pretreatment were sterile (N = 3-5). Mouse spleens, removed sterilely, were placed in RPMI-1640 at 4°C cells were teased free, and red cells were removed using separation media (Lympholyte-M, Cedarlane Laboratories, Hornby, Ontario, Canada) and centrifugation at 25°C at 500g for 20 min. Pellets were resuspended in RPMI-1640 and cells were allowed to adhere to polystyrene tubes for 2 hr at room temperature to remove macrophages. Nonadherent cells were washed three times in RPMI-1640 at 4°C at 500g for 10 min and then resuspended in RPMI-1640 with 10% fetal bovine serum (Sigma) at the appropriate dilution. Cells treated this way were found to be consistently ~2% macrophages by nonspecific esterase staining and had ~85% viability by trypan blue exclusion. Wright’s staining revealed 290% lymphocytes.

fragilis,

ABSCESS

83

FORMATION

Labeling of Mixed Splenic Lymphocytes FACS Analysis

and

Mixed splenic lymphocytes were labeled with fluorescein isothiocyanate (FIT0conjugated antibodies for measurement of B- and T-cell percentages (18). Briefly, to label B cells, lo6 mixed lymphocytes were resuspended in phosphate-buffered saline with 0.1% bovine serum albumin and 0.02% sodium azide and incubated with 50 ~1 of a 1:lO dilution of FITC-labeled goat anti-mouse IgG antibody (Cappel, West Chester, PA) at 4°C for 1 hr. Cells were washed and resuspended at lo6 cells/ml. To label T cells, lo6 mixed splenic lymphocytes were incubated with 50 ~1 of a 1:50 dilution of FITC-conjugated mouse anti-mouse Thy 1.2 antibody (clone TS, Sigma) at 4°C for 1 hr. Cells were washed and resuspended at lo6 cells/ml. Isolated T cells were labeled for analysis of CD4 and CD8 subsets. For CD4’ cells, lo6 mixed T cells were incubated with 50 ~1 a 1:lO dilution of rat monoclonal anti-mouse CD4 antibody (clone GK 1.5, a gift of Dr. Victor Engelhard, University of Virginia, Charlottesville, VA) at 37°C for 1 hr. Cells were washed, resuspended at lo6 cells/ml, and incubated with 50 ~1 of a 1:50 dilution of FITC-labeled goat anti-rat IgG antibody (Cappel) at 4°C for 1 hr. To label CD8’ cells, lo6 mixed T cells were incubated with 50 ~1 of a 1:lO dilution of mouse monoclonal IgM anti-mouse Lyt 2.2 antibody (clone AD4(15), Cedarlane) at 4°C for 1 hr. Cells were washed, resuspended at lo6 cells/ml, and incubated with 50 ~1 of a 1: 10 dilution of FITC-labeled goat anti-mouse IgM antibody (CL chain specific, Accurate Chemical, Westbury, NY) at 4°C for 1 hr. Cells were sorted in a standard fashion on a FACS machine (Becton-Dickenson, Mountain View, CA), and relative percentages of fluorescently labeled cells were derived with EPICS Cytologic Software (Coulter Corporation, Hialeah, FL). Processing Transfer

of LymphocyteslSerum Experiments

and Passive

Mixed splenic lymphocytes were separated into B and T cells by panning across anti-mouse polyvalent immunoglobulin as previously described (19). Briefly, 10 ml of a solution of 10 pgm/ml goat anti-mouse IgG and IgM immunoglobulin (Accurate Chemical) in Trisbuffered saline at pH 9.5 was incubated at 25°C for 1 hr in polystyrene petri dishes to adhere antibody. Plates were washed three times in PBS and 1% FBS and kept at 4°C until use. The 2 x lo7 mixed lymphocytes were resuspended in 3 ml PBS and 5% FBS and incubated on the plates at 4°C for 1 hr. After incubation, nonadherent cells (T cells) and adherent cells (B cells, dislodged after three gentle rinses with ice-cold medium) were washed three times and resuspended at the ap-

84

SAWYER

ET AL.

propriate concentrations in RPMI-1640 for either pas- jection of 0.5 ml 0.9% saline, and animals were resive transfer or further separation into subsets. turned to their cages and allowed oral intake of chow For enrichment of CD4’ cells, lo7 mixed washed T and water ad lib. cells were resuspended in 1 ml cytotoxicity medium Ten days after abscess induction, mice were sacri(Cedarlane) and incubated with 50 ~1 of a 1: 10 dilution ficed and all intraperitoneal abscesses 22 mm were of anti-mouse Lyt 2.2 antibody and 100 ~1 of rabbit excised, measured in greatest diameter, placed in precomplement (Low-Tox-M, Cedarlane) at 37°C for 1 hr reduced Hanks’ balanced salt solution, and weighed. to delete CD8’ cells. The resultant cells were washed Previous experiments with this model have shown that three times and resuspended at the appropriate diluabscesses <2 mm in diameter are invariably sterile (untion in RPMI-1640 at 4°C prior to passive transfer. For published data). Abscesses were minced, and the hoCD8’ enrichment, lo7 mixed washed T cells were likemogenate was serially diluted and quantitatively wise resuspended in cytotoxicity medium and incuplated aerobically on McConkey’s agar and anaerobibated with 50 11 of a 1:lO dilution anti-mouse CD4 cally on brucella agar. Surveillance culture for nonantibody and 100 ~1 of rabbit complement at 37°C for 1 gram-negative organisms was carried out intermithr to delete CD4” cells. Cells were washed three times tently via aerobically incubated blood agar plates. and resuspended in medium prior to passive transfer. Growth was evaluated 24 and 48 hr after plating. MinThe purity of isolated cells was ascertained by fluoimum detectability for bacteria was =l x lo2 CFUI rescent microscopy of cells labeled as for FACS analyabscess. sis (see above). Between 100 and 200 viable (by trypan Nonparametric variables (mortalities) were comblue exclusion) cells per sample were counted by hand. pared using x2 analysis. Multiple means were first In each case, 298% of viable cells were found to be compared using a two-way ANOVA (Fisher PLSD); if a fluorescently labeled, indicating adequate deletion of difference was detected, results were retested using an unwanted subsets. unpaired Student’s t test with a Bonferroni adjustAs indicated by experimental groups (Tables 2 and ment. Therefore, for splenic lymphocyte data, a P G 31, serum or cells were passively transferred under 0.01 was considered significant; for abscess data, with aseptic conditions to recipient mice 24 hr prior to the larger numbers of groups, a P < 0.005 was considered induction of abscesses. Either 0.15 or 0.3 ml of serum, significant. All comparisons were made to data derived obtained by centrifugation of whole blood from 1-2 from similar numbers of concurrently studied control mice at 1OOOg for 5 min in serum-separator tubes, was animals (n = 10-201, although the data reported for the transferred via intramuscular injection. Immunoglobuntreated group is for all 30 control mice surviving to ulin content was not assayed. A total of 150 ~1 of ap- sacrifice. Statistical software packages (StatWorks, propriate cell suspensions (total cells: 5 x lo6 B cells or CricketSoftware, Philadelphia, PA, and StatViewmixed T cells or 2.5 x lo6 CD4’ or CD8’ T cells) was 512+, Abacus Concepts, Calabasas, CA) were used for transferred under light halothane anesthesia by intracalculations. cardiac injection. Tables 2 and 3 describe controls and the 33 groups RESULTS studied after passive transfer. Abscess Induction,

Analysis,

Alterations in Splenic Lymphocyte by Preexposure

and Statistics

Abscesses were induced as previously described (5). Mice received intraperitoneal injections of a mixture of lo3 live E. coli, lo5 live B. fkagilis, and 50-~1 sterile fecal adjuvant solution (total volume 175 ~1). Acute volume resuscitation was given via subcutaneous in-

Subsets

The percentages of splenic lymphocytes found to be B cells (IgG+) and T cells (Thy 1.2+), and the percentage of isolated T cells found to be CD4+ cells (CD4+) and CDS’ cells (Ly 2.2+), are shown in Table 1. Preexposure to either E. co&, B. fiagilis, or both, significantly

TABLE

1

Changes in Lymphocyte Subsets after Bacterial Preexposure Bacterial preexposure

Spleens studied

% of Lymphs I&+

% of Lymphs Thy 1.2’

% of T cells CD4’

% of T cells CD8’

None E. coli B. fiagilis E. coli and B. fhgilis

13 8 8 8

45 f 1 41 f 2 38f2” 42 f 1

36k 1 50 f 1” 54 f 1” 51il”

60f 1 56f2 56 f 2 57f 1

32i 1 31fl 33 f 1 32 f 2

Note. All percentages

are f SEM, “P < 0.01 vs untreated

controls by unpaired

Student’s t test.

CDUCD8 1.9 1.8 1.7 1.9

f f f f

0.1 0.1 0.1 0.1

CD4’ LYMPHOCYTES

increased the percentage of nocytes compared to naive ward a reciprocal decline in Analysis of CD4’ and CD8’ ference among groups and CD4+/CD8’ ratios. Passive Transfer

AND ABSCESS

85

T cells among mixed splesiblings, with a trend tothe percentage of B cells. subsets showed little difno significant change in

of Serum and Mixed Lymphocytes

Mice received either 0.15 or 0.3 ml serum or 103, 105, or lo7 mixed lymphocytes from either naive or preexposed siblings (N = 3 to 15 survivors per group, Table 2). Mortality was not different between groups. The only consistent differences in bacterial composition were found in groups receiving serum from siblings preexposed to B. fiagilis, either alone or in combination with E. coli. In those groups, significantly more B. fragilis were cultured from each abscess, implying a possible humoral immune response to B. fiagilis. In no group were organisms other than E. coli and B. fiagilis cultured. The number of abscesses formed in control animals and the mixed lymphocyte transfer groups are shown in Fig. 1. The passive transfer of serum did not change the number of abscesses formed. The passive transfer of lo7 lymphocytes, but not lo3 or lo5 lymphocytes, from preexposed mice increased the subsequent number of abscesses in naive animals. Lymphocytes transferred from naive siblings had no effect on abscess formation. Passive Transfer

of B and T Lymphocytes

A total of 5 x lo6 enriched B or T cells from naive or preexposed animals were passively transferred to na-

Group

FORMATION

Size, Mortality,

Abscess

Size, and Bacteria

Amount, source, and substance transferred

Mice injected

None 0.15 ml naive serum 0.15 ml EC serum 0.15 ml BF serum 0.15 ml ECIBF serum 0.30 ml naive serum 0.30 ml EC serum 0.30 ml BF serum 0.30 ml ECIBF serum lo3 EC lymphs lo3 BF lymphs lo5 EC lymphs lo6 BF lymphs lo7 naive lymphs lo7 EC lymphs lo7 BF lymphs lo7 ECIBF lymphs

37 5 5 5 5 16 16 16 16 16 16 16 16 18 22 16 17

Lymphocytes

(loglo)

FIG. 1. Number of abscesses formed (+SEM) after standard mixed intraperitoneal infection in groups receiving passive transfer of 0, 103, 105, or lo7 mixed splenic lymphocytes from naive siblings or mice preexposed to E. coli, B. fro&is, or both. *P < 0.005 vs untreated controls and mice receiving lo7 naive lymphocytes, by unpaired Student’s t test.

ive siblings (iV = 6 to 9 survivors per group, Table 3). Mortality was not significantly different among groups. Bacterial composition was dependent on treatment, with animals receiving B cells from donors preexposed to B. fragilis, either with or without E. co& having significantly higher numbers of anaerobes cultured per abscess, as had been seen with animals receiving serum from the same sources. The transfer of preexposed T cells, but neither preexposed B cells nor

TABLE 2 Cultured for Controls Deaths by 10 days (o/o)

Transferred

and Mice Receiving

Mean abscess size (mm)

7 (19) 2 (40)

l(20) l(20)

l(20) 3 (19) 3 (19) 5 (31) l(6) 3 (19) 3 (19) 3 (19) 4 (25) 6 (33) 7 (32) 4 (25) 6 (35)

Note. Results are it SEM. EC, E. coli; BF, B. frugilis; CFU, log,, colony-forming D P < 0.005 versus untreated controls by unpaired Student’s t test.

4.4 4.1 5.0 5.5 5.3 5.0 4.5 5.4 4.4 4.2 5.0 4.4 4.3 4.8 3.9 4.2 4.4

f 0.2 f 0.4 f 0.8 + 1.1 + 0.5 f 0.4 f 0.2 5 0.5 + 0.4 f 0.3 f 0.4 It 0.4 f 0.3 It 0.3 f 0.2 It 0.3 f 0.3

units/abscess.

Serum

CFU E. coli 7.0 7.0 7.0 7.3 7.0 6.8 6.8 7.0 7.0 6.9 7.0 6.9 6.7 6.8 6.6 6.6 6.7

I!z0.2 * 0.5 f 0.7 rt 1.0 f 0.3 f 0.1 f 0.2 f 0.3 * 0.3 + 0.4 * 0.4 ic 0.3 It 0.2 It 0.1 L- 0.6 f 0.2 f 0.1

or Mixed

Lymphocytes CFU B. fkgilis 7.0 7.0 7.0 7.4 7.5 7.0 7.0 7.3 7.4 7.2 7.3 7.0 7.0 7.0 6.9 6.7 7.0

f + f f f f * f * + f f f f k f *

0.2 0.2 0.3 0.2” 0.2” 0.2 0.2 0.1” 0.2” 0.3 0.5 0.3 0.3 0.3 0.2 0.2 0.2

SAWYER

86

ET AL,.

TABLE 3 Abscess Size, and Bacteria Cultured for Controls and Mice Receiving B Cells, T Cells, or T Cell Subsets

Group Size, Mortality,

Deaths by 10 days (%)

Source and type of cells transferred

Mice injected

None Naive B cells EC B cells BF B cells ECIBF B cells Naive T cells EC T cells BFT cells EC/ BF T cells Naive CD4+ T cells EC CD4’ T cells BF CD4’ T cells ECIBF CD4’T cells Naive CD8’ T cells EC CDS’ T cells BF CD8’ T cells ECIBF CD8’ T cells

37 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Mean abscess size (mm)

7 (19) l(10)

4.4 4.1 4.6 4.3 4.2 4.9 4.7 4.2 4.5 4.5 4.4 4.5 4.3 4.3 5.5 4.1 4.5

2 (20) 2 (20) 3 (30) 4 (40)

2 (20) 3 (30) 3 (30) l(10) 0 (0)

2 (20) l(10) 4 (40) 2 (20) 2 (20)

2 (20)

Note. Results are f SEM. EC, E. coli; BF, B. fragilis; CFU, log,, colony-forming D P < 0.005 versus untreated controls by unpaired Student’s t test.

either cell type from naive animals, significantly creased the number of abscesses (Fig. 2). Passive Transfer

in-

of CD4’ and CD8’ T Lymphocytes

2.5 x lo6 viable CD4’ T cells or CD8’ T cells were passively transferred. The group size, mortality after abscess induction, mean abscess size, and bacterial composition of abscesses are shown in Table 3. As beRmor

f k f k k * k t 5 k f * f f f f f

log,, CFU

0.4 0.3 0.5 0.4 0.3 0.5 0.4 0.4 0.3 0.2 0.3 0.3 0.3 0.4 0.6” 0.5 0.7

7.0 7.0 6.7 7.0 7.1 7.0 6.8 6.8 7.0 6.7 6.7 7.0 6.3 7.0 6.7 7.0 7.0

Naive

q

E. co/i

H

6. ffagilis

Lymphocyte

7.0 f 0.2 7.1 f 0.3 7.0 zk 0.3 7.5 k 0.2” 7.4 f 0.2” 7.1 f 0.5 6.9 310.2 7.0 It 0.3 7.1+ 0.4 7.0 k 0.2 7.0 ?r 0.3 7.1 f 0.3 7.0 f 0.2 7.1* 0.4 7.0 III 0.4 7.0 * 0.2 7.1 zk 0.3

units/abscess.

fore, passive transfer of these T cells did not significantly alter any of these parameters, with the exceptions of abscess size in mice receiving CD8’ cells from E. coli preexposed siblings. The meaning of this isolated finding is unknown. The number of abscesses in these groups is shown in Fig. 3. Transfer of preexposed donor cells enriched for CD4’ cells, but not CD8’ cells, reproduced the augr ore-exow *

* * T

s 06llS

None

B. fragilis

f 0.2 + 0.4 f 0.2 f 0.3 It 0.6 f 0.4 * 0.1 It 0.2 It 0.4 f 0.2 f 0.2 f 0.4 f 0.1 lb 0.5 + 0.1 * 0.4 + 0.3

Dwxmmm

n

log,, CFU

E. coli

Subset

**

* J7‘,‘,

TT

T cells

ths

Transferred

FIG. 2. Number of abscesses formed (ItSEM) after standard mixed intraperitoneal infection in groups receiving passive transfer of 5 x lo6 B cells or 5 x lo6 T cells from naive siblings or mice preexposed to E. coli, B. fiagilis, or both. *P < 0.005 vs untreated controls and mice receiving naive T lymphocytes, by unpaired Student’s t test.

CD4+

n

Naive

El

E. co/i

El

8. ffagi/is

q E.co/m. fragifis

CDS+

T Cell Subset Transferred FIG. 3. Number of abscesses formed (LSEM) after standard mixed intraperitoneal infection in groups receiving passive transfer of 2.5 x lo6 CD4’ or 2.5 x lo6 CD8’ T cells from naive siblings or mice preexposed to E. coli, B. fragilis, or both. *P < 0.005 vs untreated controls and mice receiving naive CD4’ T cells, by unpaired Student’s t test.

CD4’ LYMPHOCYTES

AND ABSCESS

mentation in abscess number seen previously. CD4’ or CD8’ T lymphocytes from naive siblings did not have this effect. DISCUSSION

In order to study the immunological effects of transient bacterial infections, like those seen clinically with wound infections, line sepsis, urinary tract infections, etc., on subsequent, severe infections, we have developed a model of mixed E. colilB. fragilis peritonitis and abscess formation in which preexposure to live bacteria results in a greater number of intraabdominal abscesses (5-7). It must be emphasized that this complex system is designed neither to produce immunity nor to reproduce the severe immunological dysfunction seen with trauma, burns, the systemic inflammatory response syndrome (SIRS), or multisystem organ failure (l-4). In this sense, the model is unique in its demonstration of significant immunological changes after an apparently trivial and innocuous bacterial infection. By passive transfer experiments, the data presented here establish the importance of the cellular immune response, particularly CD4’ T cells, in this model. Although some element of humoral response was noted, as indicated by increases in numbers of recoverable B. fragilis after the transfer of serum or B cells from B. fragilis-exposed donors, this effect was weak and of unknown significance, since B. fragilis preexposure alone does not consistently give a similar result (5, 6). In general, whether this response to a transient exposure to bacteria is beneficial or harmful to the host is debatable, since differences in overall mortality were not observed. The increase in number of abscesses could conceivably be viewed as either improved containment or deficient killing of bacteria. The fact that CD4’ cells are important in this system does not imply enhanced immune activity either, since a Thl subset response would be expected to promote cellular immune function, while a Th2 dominant response could promote humoral- and inhibit cell-mediated immunity (20, 21). These results further imply a central pathophysiologic role for CD4’ lymphocytes in the development of abscesses, a finding not predicted by earlier studies showing CD4’8+/CDB’-mediated immunity to monomicrobial B. fragilis abscesses (8-17). Several key differences between the models used, however, could explain the disparate results,. First, the studies may represent opposite ends of the same spectrum: one set of data examining CD4’8”/CD8’-driven immunity; the other, CD4’-driven abscess formation. Second, there are certainly dissimilar host immune responses to purified, inert antigen and an actual bacterial infection. Third, the bacteriological nature of the peritonitis induced, monomicrobial versus polymicrobial, is very

FORMATION

a7

different, particularly in light of the well-described synergy between E. coli and B. fragilis in mixed infections (22, 23). Attempts to define the mechanisms involved in the described CD4+ lymphocyte response are ongoing. At a regulatory level, this model exhibits MHC restriction, since preliminary data indicate transfer of preexposed donor lymphocytes across mouse strains does not reproduce the same changes in abscess number (24). Although the relative contributions of class I and class II recognition are not known, these findings are consistent with a classic CD4+/T helper-class II interaction. At the effector level, the upregulation of macrophage procoagulant activity (PCA) may be important, since preliminary data suggest that PCA is critical to abscess formation in this model of peritonitis (25); there is a correlation between other nosocomial pathogens’ ability to induce PCA and increase abscess formation (7); and lymphocytes from preexposed donors can directly upregulate PCA in response to a standard stimulus, though in a non-MHC-restricted fashion (22). Experiments to determine the cytokine profile and T helper subset (Thl versus Th2) producing these effects are underway. The data presented reemphasize the marked effect apparently trivial infections can have on the outcome of subsequent, more serious infection, and begin to define a mechanism for these changes. This outcome is dependent on CD4’ T cells and underlines the potential importance of the cell-mediated immune system in acute and chronic bacterial infections. REFERENCES A. J., and Mannick, J. A., Lymphocyte function in the ill surgery patient. Surg. Clin. North Am. 63, 245-261,

1. M&vine, critically 1983. J. A., Impairment of T-cell activation in 2. Teodorczyk-Injeyen, burn patients: A possible mechanism of thermal injury induced immunosuppression. Clin. Exp. Immunol. 65, 570-581, 1986. 3. Richards, P. S., and Saba, T. M., Alterations of fibronectin and reticuloendothelial phagocytic function during adaptation to experimental shock. Circ. Shock 10, 189-198, 1983. 4. Almdahl, S. M., and Osterud, B., Experimental gram-negative peritonitis: Decreased thromboplastin activity in organs with a simultaneous rise of thromboplastin in blood monocytes and peritoneal macrophages. Res. Exp. Med. 186,317-324, 1986. 5. Sawyer, R. G., Adams, R. B., Spengler, M. D., and Pruett, T. L., Pre-exposure of the peritoneum to live bacteria increases later mixed intraabdominal abscess formation and alters mortality. J. Infect. Dis. 163, 664-667, 1991. 6. Sawyer, R. G., Adams, R. B., Spengler, M. D., and Pruett, T. L., Transient and distant infections alter later intraperitoneal abscess formation. Arch. Surg. 126, 164-168, 1991. 7. Sawyer, R. G., and Pruett, T. L., Nosocomial pathogens: Effect of transient bacteremia on later intraperitoneal abscess formation correlates with in uitro upregulation of macrophage procoagulant activity. Crit. Care Med., in press. 8. Kasper, D. L., Onderdonk, A. B., Crabb, J., and Bartlett, J. G., Protective efficacy of immunization with capsular antigen

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ET AL.

feet. Dis.

9.

10.

11.

12.

13.

15.

16.

Received January

20, 1995; accepted with revision May 23, 1995

17.

18.

19.

20.

21.

22.

23.

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