essing and presentation, and the down regulation of immune responses through the elimination of antigen.

Transcription

1 INFECTION AND IMMUNITY, June 1987, p /87/ $02.00/0 Copyright C) 1987, American Society for Microbiology Vol. 55, No. 6 An Early Response to Lipopolysaccharide Is the Elicitation of Macrophages Specialized for Antigen Degradation with Negative Regulatory Effects on the Induction of Specific Immune Responses CHRISTOPHER W. CLUFF AND H. KIRK ZIEGLER* Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia Received 6 October 1986/Accepted 17 February 1987 The ability of macrophages to catabolize antigens is relevant both as a means to process complex antigens before presentation to T cells and as a way to down-regulate immune responses by destroying the antigenicity of polypeptides. With these considerations in mind, we investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of 1251-labeled surface components of heat-killed Listeria monocytogenes after their uptake by macrophages. We compared the catabolic activity of macrophages from peritoneal exudates of mice injected intraperitoneally with saline or LPS and found that LPS-elicited macrophages displayed a greatly enhanced (threefold) rate of catabolism. This increase in catabolic activity peaked 3 days after LPS injection and slowly declined thereafter, approaching a base-line level after 3 weeks. The enhancement of catabolic activity was under Lps gene control. Macrophages that were elicited 3 days after intraperitoneal injection of LPS rapidly destroyed the antigenicity of bacterial antigens, expressed low levels of Ia molecules, and processed and presented antigen slowly when tested as antigen-presenting cells in vitro. We also showed that an injection of LPS before infection with L. monocytogenes resulted in diminished development of T-cell reactivity to this organism. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions, with negative regulatory effects on the induction of specific immune responses. It has been firmly established that antigen-induced stimulation of resting T lymphocytes requires the participation of accessory cells, termed antigen-presenting cells (APCs) (10, 17). Macrophages have been shown to function as APCs in vitro. It appears that particulate antigens, such as intact bacteria, must first be ingested by the APCs and processed to a form that can associate properly with Ia molecules before stimulation of T cells can occur. This processing event was first demonstrated definitively by Ziegler and Unanue (27, 28), who showed that although macrophages can take up bacteria quickly (within 5 min), antigen presentation requires a 30- to 60-min period of antigen-macrophage interaction. This postbinding, antigen-handling event required for antigen presentation was termed antigen processing, which was characterized as a temperature-dependent and energy-requiring event that is inhibited by lysosomotropic agents such as chloroquine and ammonia or by fixation with paraformaldehyde. The kinetics of antigen catabolism correlates with antigen processing, and both catabolism and processing are inhibited by similar conditions. Thus, catabolism appears to be a requisite event for the presentation of complex antigens (27, 28). However, the catabolic breakdown and elimination of antigen by macrophages are also relevant mechanisms for the down regulation of the immune response. The catabolism of ingested proteins by macrophages has been shown to be fairly complete, with most material released in the form of free amino acids or weakly antigenic, small-molecularweight peptides (1, 19). It appears, then, that antigen catabolism is required for both the induction of specific immune responses, via proc- * Corresponding author. essing and presentation, and the down regulation of immune responses through the elimination of antigen. Lipopolysaccharide (LPS) is a biologically active molecule present as an integral part of the cell wall of gramnegative bacteria. Intraperitoneal (i.p.) administration of LPS has been shown to elicit a population of macrophages that expresses increased amounts of the enzymes acid phosphatase,,b-glucuronidase, and cathepsin (3) and releases greater amounts of superoxide anion (02) upon stimulation with phorbol myristate acetate in vitro (5). Macrophages elicited early (3 days) after an i.p. injection of LPS have also been shown to express low levels of Ia molecules (25). Galleli et al. (8) have demonstrated an increased resistance to infection but a diminished immune response to later challenge in mice injected with LPS before challenge with Listeria monocytogenes. Recently, we found that an i.p. injection of LPS elicits a population of macrophages that is highly catabolic. Because catabolic activity is required for processing and presentation of complex antigens, it seemed likely that the increased catabolic activity observed with LPS-elicited macrophages might enhance the rate at which these cells process antigen. Despite this expectation, we have found that these highly catabolic macrophages process antigen slowly. Using heat-killed L. monocytogenes as antigen, we have investigated the effect of LPS on the catabolic activity of macrophages and the role that this activity may play in the elicitation of specific immune responses and resistance to bacterial infection MATERIALS AND METHODS Mice. C3HeB/FeJ and C3HeB/HeJ female mice at 6 to 8 weeks of age (Jackson Laboratory, Bar Harbor, Maine) were used.

2 VOL. 55, 1987 Reagents. LPS used in these experiments was prepared from Salmonella minnesota R595 as described by Galanos et al. (7). LPS was generously supplied by David Morrison and purchased from Ribi Immunochem (Hamilton, Mont.). LPS concentrations were determined by weighing and confirmed with a 2-keto-3-deoxyoctulosonate assay (21). LPS was diluted in phosphate-buffered saline (PBS) and sterilized by being boiled for 15 min. Sterility was confirmed by 24-h incubation at 37 C on brain heart infusion agar plates. Media. Peritoneal lavage was performed with Hanks balanced salt solution containing 0.06% bovine serum albumin, 10 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 2 mm L-glutamine, 50 U of penicillin per ml, 50 p,g of streptomycin per ml, and 10 U of heparin per ml. This medium, minus the heparin, was used for cell washing procedures (referred to as wash medium). For cell culture, RPMI 1640 containing 5% fetal calf serum, 10 mm HEPES buffer, 2 mm L-glutamine, 50 U of penicillin per ml, 50 jig of streptomycin per ml, 0.075% sodium bicarbonate, and 0.5 mm sodium pyruvate was used. L. monocytogenes. The preparation of L. monocytogenes has been previously described (5). Live bacteria (104 i.p.) were used to immunize mice, and heat-killed organisms were used as antigen in vitro. Preparation of SLP. Soluble listerial proteins (SLP) were prepared from freshly expanded and washed live bacteria by sonication (six 5-min cycles, 30 W, 4 C), ultracentrifugation (100,000 x g, 60 min), and fractionation of the soluble material by isopycnic gradient centrifugation (37% cesium chloride [wt/wt], SW50.1 rotor, 150,000 x g, 70 h) (24). Material banding at a density of 1.3 g/ml was pooled, dialyzed against PBS, assayed for protein content by the method of Lowry et al. (12), and stored frozen. T cells and macrophages. The preparation of cell populations has been previously described (5, 6, 25-27). Briefly, T cells were purified from the peritoneal exudates of mice infected i.p. with 1 x 104 to 5 x 104 live L. monocytogenes cells. After 1 week, the mice received an i.p. injection of 10% proteose peptone (1.5 ml) and were sacrificed 3 days later. T-cell enrichment, accomplished by the removal of cells adherent to tissue culture dishes and nylon wool, routinely resulted in a more than 95% Thy-1.2-positive lymphocyte population containing less than 0.5% macrophages. To isolate macrophage populations, peritoneal exudate cells (PECs) were harvested as described above and were incubated (2 h at 37 C) in tissue culture vessels to allow macrophage adherence. The nonadherent cells were then removed by washing. Macrophage fixation procedure. Macrophages were washed twice with Hanks balanced salt solution and either fixed with 0.05% glutaraldehyde in PBS (200,ul) or sham fixed with PBS. After 2 min, the glutaraldehyde or PBS was removed and 2.19% glycine (200,ul) was added to each well. The macrophages were then washed twice with wash medium and twice with cell culture medium. After a 5-min incubation at 37 C, the cells were washed once more with cell culture medium. Assay for antigen presentation. Two experimental protocols were used: the antigen uptake and the antigen processing protocols. In the uptake protocol, macrophages (3 x 105 PECs per well) were pretreated with antigen for 30 to 360 min (pretreatment period) and then washed before addition of the T cells. T cells (3 x 105 per well) and macrophages were cultured for 18 to 24 h (culture period). Because macrophages were able to process during the culture period LPS-ELICITED MACROPHAGES 1347 the antigens that had been taken up during the pretreatment period, the degree of antigen-specific T-cell activation reflected the amount of antigen taken up by the macrophages before washing. The antigen processing protocol was identical, except that the macrophages were fixed with glutaraldehyde before the addition of T cells. Since no processing could occur after fixation, the degree of antigen-specific T-cell activation observed was directly related to the amount of antigen taken up and appropriately processed during the pretreatment period. A portion of supernatant (20 [li) from each macrophage-tcell coculture was incubated (48 h) with 106 normal thymocytes. Tritiated thymidine (25,ul of 25 p,ci/ml) was then added to each well. The thymocytes were harvested 16 to 20 h later, and thymidine incorporation was measured by standard scintillation techniques. The thymocyte proliferation (counts per minute of [3H]thymidine incorporation) that resulted from culture supernatants of macrophages and antigen (no T cells) was subtracted from the values obtained with supernatants of macrophages, antigen, and T cells. This value (A) represents a measurement of macrophage-dependent, antigen-specific T-cell activation. Similar results were obtained when supernatants were assayed with the IL- 2/BSF1-dependent HT-2 cell line. Determination of catabolic rates. (i) Kinetics protocol. The catabolic activity of the various macrophage populations was measured by two protocols. The kinetics of antigen catabolism by macrophages was determined by following the fate of radiolabeled heat-killed L. monocytogenes after uptake. These cells were iodinated as previously described (9, 27) (1 to 2 cpm per bacterium). Macrophages were purified by adherence (3 x 105 PECs per well in 48-well plates). The radiolabeled heat-killed L. monocytogenes (105 cpm per well) were added to the macrophage monolayers, and the plates were centrifuged (2,000 rpm, 5 min) to speed adherence. The plates were incubated at 37 C for 5 min to allow antigen uptake, washed three times to remove unbound heat-killed L. monocytogenes, and reconstituted with culture medium (200,ul per well). The plates were incubated at 37 C for various periods of time, after which supernatants and lysates were collected. Lysates were collected by adding 0.5 ml of 0.5% Triton X-100 to each well, incubating the preparation for 5 min at 37 C, agitating it vigorously with a Pasteur pipette, and transferring it to a 5-ml test tube. This procedure was repeated to ensure complete removal of the macrophages. An equal volume of 20% trichloroacetic acid (TCA) was added to the supernatants and lysates, which were then vortexed and cooled to 4 C for at least 30 min. The precipitate was separated from the soluble material by centrifugation (3,000 rpm, 20 min), and the amount of 1251 in each sample was determined with a gamma counter. Thus, the sum of the 125I-labeled material in each portion equaled the total amount of _251-labeled heat-killed L. monocytogenes bound by each macrophage population. We could then determine the proportion of radiolabel that was released or retained as TCA-soluble or -precipitable material at each time point. (ii) Dose-response protocol. By assaying over a broad range of antigen concentrations, we could compare the catabolic activities of the various macrophages despite differences in the amount of antigen taken up by each population. Macrophage monolayers were prepared as before. Antigens were introduced by adding to each well 100,u1 of culture medium containing 3.3 x 107, 1.1 X 107, or 3.4 x 106 heat-killed L. monocytogenes, along with 100,ul of culture medium containing I05 cpm of '251-labeled heat-killed L. monocytogenes.

3 1348 CLUFF AND ZIEGLER INFECT. IMMUN. A 70-.* 60-.:' PERCENTAGE OF TOTAL CPM 40-. BOUND 30-. g 20-./a. 10i 80 B 70- A, TIME (MINUTES) PERCENTAGE OF TOTAL CPM 40- BOUND El r TIME (MINUTES) FIG. 1. Kinetics of antigen catabolism. The kinetics by which 1251-labe!ed heat-killed L. monocytogenes cells were catabolized by 7-day saline-elicited (resident) macrophages (A) or 7-day LPS-elicited macrophages (B) was determined as described in Materials and Methods. The amount of 1251 in each portion was divided by the amount bound so as to reveal the proportion released or retained as TCA-soluble (supernatant [+] or lysate [x]) or TCA-precipitable (supernatant [rl] or lysate [*]) material after a given period of time. Values presented are means of duplicate wells (deviation from the mean did not exceed 10% of the value). The plates were centrifuged, washed, and reconstituted with culture medium as before. The plates were incubated for 1 h, after which the culture supernatant in each well was removed and replaced with fresh medium. This procedure was repeated after 4- and 24-h incubations. The macrophage lysates were collected after the final time point. The supernatants and lysates were then subjected to TCA precipitation as before, and the amount of 1251 in the soluble and precipitable portions of each was determined. By knowing the specific activity of the trace-labeled heat-killed L. monocytogenes preparations added, the total counts per minute bound, and the counts per minute released as TCA-soluble material, we could calculate the total heat-killed L. monocytogenes bound and number catabolized. Antigen destruction by macrophages. PECs from mice injected 3 days before with either 1,ug of LPS in 1 ml of PBS (for LPS-elicited macrophages) or 1 ml of PBS (for resident macrophages) were harvested as described and plated at 3 x 105 cells per well. Macrophages were purified by adherence, and culture medium or heat-killed L. monocytogenes (200 RI of 2 x 108 cells per ml in culture medium) were subsequently added. The heat-killed cells were also cultured alone as a control. The plates were centrifuged at 2,000 rpm for 5 min and incubated at 37 C for 5 min to facilitate antigen uptake. Unbound heat-killed L. monocytogenes cells were removed from the macrophage cultures by three washings, and the plates were incubated at 37 C for 0, 8, and 24 h. At these times, the supernatants from the appropriate wells were removed and 0.5 ml of sterile H20 was added. The plates were returned to the incubator for 30 min. The hypotonic lysate was removed and added to the supernatant. The wells were washed once more with 0.5 ml of sterile H20, which

4 VOL. 55, 1987 LPS-ELICITED MACROPHAGES NUMBER OF HKLM 4 (MILLION) CATABOLIZED NUMBER OF HKLM (MILLION) BOUND FIG. 2. Augmented catabolic activity of LPS-elicited macrophages. The indicated populations of macrophages were assayed for catabolic activity by the dose-response protocol as described in Materials and Methods. Data are for resident macrophages (+) and for LPS-elicited macrophages after 3 (*), 7 (El), 14 (x), and 21 (-) days. HKLM, Heat-killed L. monocytogenes. was also added to the supernatant. Hanks balanced salt solution (10x solution) was added to each of the supernatant-lysate mixtures to return them to physiological tonicity. These preparations were then tested for antigenic activity by adding 20 [li of various dilutions (in culture medium) to 106 PECs (from L. monocytogenes-immune mice containing antigen-specific T cells and macrophages) in microtiter wells (100 pll). Supernatants were collected after 24 h at 37 C and evaluated for lymphokine content as described above. Solid-phase radioimmunoassay for detection of macrophage surface Ia antigens. This technique has been previously described in detail (16). All incubations were for 60 min at 20 C, and the intervening three washes and reagent diluations were performed with PBS containing 0.1 mg of bovine serum albumin per ml. In step 1, fixed macrophages were incubated with 50,ul of 1% bovine serum albumin per well to reduce nonspecific binding. Incubation 2 was with 25,ul of anti-iak (102.16, 1:2 dilution) or control immunoglobulin G2b (IgG2b) myeloma protein (5 jig/ml) per well. Incubation 3 was with 25,ul of a 1:300 dilution of rabbit anti-mouse IgGl, IgG2, IgG3, and IgM (Litton Bionetics, Kensington, Md.) per well. Incubation 4 was with 25,ul of 125I-labeled Staphylococcus aureus protein A per well. Protein A was labeled with 1251 (New England Nuclear Corp., Boston, Mass.) by a chloramine-t method (24) and stored at 4 C for up to 4 weeks (specific activity, 0.5 to 1.0 uci/,lg). Approximately 100,000 cpm per well was used. After incubation 4, the plate was washed three times and cut with a hot wire, and radioactivity was determined by a gamma counter. Experimental values are represented as the average of triplicates with control values subtracted (Acpm). The number of adherent macrophages per well was confirmed by random field counting and determination of total protein (16). RESULTS The catabolic activity expressed by resident or 7-day LPS-elicited macrophages was first determined by following the fate of radiolabeled heat-killed L. monocytogenes after uptake by each population of cells. The radiolabeled heatkilled cells (105 cpm per well) were added to the macrophage monolayers, the unbound heat-killed cells were removed, and the supernatants and lysates were collected after various periods of time and subjected to TCA precipitation (as described in Materials and Methods for the kinetics protocol). Most of the radiolabel taken up by the macrophages was eventually released in a TCA-soluble form (small molecular weight) (Fig. 1). The kinetics of catabolism shown by each macrophage population were compared by noting the time points at which the amount of radiolabel released in a TCA-soluble form equaled the amount of label that remained associated with the macrophages in a TCA-precipitable form. It was apparent from this analysis that LPS-elicited macrophages could degrade antigen to a TCA-soluble form faster than resident macrophages could (Fig. 1). Whereas LPS-elicited macrophages released as much radiolabel in a TCA-soluble form as they retained as precipitable material in 3 h, resident macrophages required 6 h to degrade the bacteria to the same extent. Because the LPS-elicited macrophages took up on the average only -85% as many heat-killed L. monocytogenes cells as did the resident macrophages (data not shown), it was necessary to determine the catabolic activity of these macrophage populations over a range of antigen concentrations. Macrophages derived from the PECs of mice injected 3, 7, 14, or 21 days before with either 1 jig of LPS or 3 days before with saline (resident macrophages) were tested for catabolic activities as described in Materials and Methods (dose-response protocol). This approach was used to compare the catabolic activities of LPS-elicited and resident macrophages. A plot of the heat-killed L. monocytogenes cells bound as a function of those degraded and released allowed the comparison of catabolic activities at equal amounts of bound antigen. It is apparent from the data presented in Fig. 2 that LPS-elicited macrophages exhibited considerably higher catabolic activity than did resident macrophages. This increase in catabolic activity peaked by day 3 and then diminished reaching control levels by day 21. In five experiments performed, the catabolic activity of macrophages harvested from mice injected i.p. 3 days before with LPS was consis-

5 1350 CLUFF AND ZIEGLER INFECT. IMMUN. 7T NUMBER OF HKLM 4- (MILLION) CATABOLIZED 31I. 1+ O ib NUMBER OF HKLM (MILLION) BOUND FIG. 3. Lack of increased catabolic activity in macrophages of mice unresponsive to LPS. The indicated macrophage populations were assayed for catabolic activity by the dose-response protocol as described in Materials and Methods. Data are for C3HeB/HeJ resident (*) and LPS-elicited (x) macrophages and C3HeB/FeJ resident (+) and LPS-elicited (E) macrophages. HKLM, Heat-killed L. monocytogenes. tently at least threefold higher than that of resident macrophages with high numbers of bound heat-killed L. monocytogenes (>5 million). Because'the macrophages harvested at day 3 displayed the highest level of catabolic activity, they were used in all subsequent experiments. To determine whether the observed LPS-induced increase in macrophage catabolic activity was under genetic control, the LPS nonresponder mouse strain, C3HeB/HeJ, was used. Resident and LPS-elicited macrophages were harvested from appropriately treated C3HeB/HeJ and C3HeB/FeJ mice and assayed for catabolic activity. The inability of LPS to induce increased catabolic activity in macrophages from A CPM C3HeB/HeJ mice (Fig. 3) indicates that this response is under Lps gene control. Because LPS-elicited macrophages exhibited high catabolic activity, it was of interest to compare the abilities of resident and LPS-elicited macrophages to process and present antigen to T cells. The kinetics of antigen uptake and antigen processing by the two macrophage populations were quantitated by using the protocols described in Materials and Methods. The results (Fig. 4) indicated that, early after antigen interaction, the LPS-elicited macrophages processed heat-killed L. monocytogenes cells slowly. Whereas resident macrophages are able to process these cells and effectively TIME (HOURS) TIME (HOURS) FIG. 4. Protocols. Antigen uptake (PBS) and antigen processing (FIX) protocols were used as described in Materials and Methods. Heat-killed L. monocytogenes cells (107 per ml) were incubated with resident (A) and LPS-elicited (B) macrophage populations (derived from 3 x 105 PEC) for 0.5, 1.5, 3, or 6 h. At these times the macrophages were washed and either fixed with 0.05% glutaraldehyde (FIX) or sham fixed with PBS. L. monocytogenes-specific T cells (3 x 105 per well) were added, and the supernatants were collected 24 h later and tested for lymphokine activity. T-cell-dependent lymphokine activity is expressed as A cpm of [3H]thymidine incorporation. Values presented are means of triplicate wells ± standard deviations.

6 VOL. 55, 1987 LPS-ELICITED MACROPHAGES PERCENTAGE OF ANTIGENIC ACTIVIlY RELATIVE TO TIME ZERO G TIME (HOURS) FIG. 5. Destruction of antigenic activity of heat-killed L. monocytogenes during incubation with LPS-elicited or resident macrophages. The heat-killed cells (200 RI of 2 x 108/ml) were centrifuged onto the indicated macrophage populations, and the hypotonic lysates, containing heat-killed L. mono. togenes cells in various stages of degradation, were collected after 8 and 24 h. The lysates (20 p.l of a 1:100 dilution) were tested for residual antigenic activity by adding them to 106 peptone-elicited, L. monocytogenes-primed PECs (in 200,ul of culture medium) and measuring resultant lymphokine production as described in Materials and Methods. Lymphokine production by PECs in the absence of antigen was subtracted from that of PECs plus antigen (Acpm). The Avcpm from PECs plus 8- and 24-h lysates was divided by the Acpm from PECs plus time zero lysates to determine the percentage of antigenic activity relative to time zero. Values presented are means of triplicate wells ± standard deviations. present the relevant antigenic determinants to T cells within 3 h, the LPS-elicited macrophages were not able to process heat-killed L. monocytogenes to a form recognizable to T cells in the same period of time. LPS-elicited macrophages may process heat-killed L. monocytogenes slowly because they degrade the bacteria to a form that is unable to effectively associate with the Ia molecules on the macrophage cell surface and to stimulate T cells. This possibility was approached experimentally by determining the ability of these macrophages to reduce antigen to a form unrecognizable by specific T cells when presented by fresh APCs. Such antigen destruction by macrophages was analyzed as described in Materials and Methods. The results of a representative experiment are presented in Fig. 5. After 24 h, the lysate of resident macrophages retained more than 95% of the antigenic activity associated with a lysate obtained immediately after MACROPHAGE POPULATION 3 DAY SAUNE-ELICITED MACS 3 DAY LPS-EUCITED MACS introduction of heat-killed L. monocytogenes (time zero). However, in the same time period, LPS-elicited macrophages reduced nearly all (>95%) of the antigen they ingested to a nonantigenic form. It is also possible that LPS-elicited macrophages process and present antigen slowly because they possess a low density of Ia molecules on their cell surfaces. A radioimmunoassay was used to quantify the Ia expression by the various macrophage populations. Macrophages harvested from mice that were injected i.p. with LPS 3 days before harvest expressed less Ia than did resident macrophages, L. monocytogenes-elicited macrophages, or macrophages har- TREATMENT WITH: NO LISTERIA SALINE PRIOR TO LISTERIA LPS PRIOR TO LISTERIA -*- LPS-ELICITED MACROPHAGES -e- RESIDENT MACROPHAGES. RESPONSE TO HKLM _ RESPONSE TO i : r ~~SLP L- I: 7 DAY LPS-EUCITED MACS 3 DAY USTERIA-EUCITED MACS oo IA (DELTA CPM) FIG. 6. Comparison of Ia density for various macrophage populations. Mice were injected i.p. with 2.5 x 103 live L. monocytogenes cells in 1 ml of saline, 1,ug of LPS in 1 ml of saline, or 1 ml of saline. PECs were harvested on the indicated day, plated at 1.5 x 105 per well, and assayed for Iak content by radioimmunoassay as described in Materials and Methods. Values presented are means of triplicate wells + standard deviations DELTA CPM FIG. 7. Decreased response to L. monocytogenes with preinfection exposure to LPS. Mice were injected i.p. with either 1 ml of PBS or 1 pug of LPS in 1 ml of saline 1 day before an i.p. injection of 2.5 x 105 live L. monocytogenes organisms. Mice injected with nothing were used as a negative control (no development of acquired immunity). After 4 weeks, the mice were injected i.p. with 1.5 ml of 10% proteose peptone, and the PECs were collected 3 days later. The PECs (106 per well in 200 pj) were incubated with heat-killed L. monocytogenes (107/ml) or SLP (50 plg/ml) for 24 h. The supernatants were then tested for lymphokine activity as described in Materials and Methods. Values presented are means of triplicate wells + standard deviations.

7 1352 CLUFF AND ZIEGLER vested from mice injected with LPS 7 days before analysis (Fig. 6). From the experiments described, it appears that LPS elicits a population of macrophages with low Ia expression and high catabolic activity. Galleli et al. (8) have shown that an i.p. injection of LPS 24 h before bacterial challenge enhances the primary resistance of the mice to infection, yet results in the generation of poor specific immunity. We have confirmed these results through experiments aimed at determining whether injection of LPS in vivo affects the ability of mice to generate L. monocytogenes-specific T cells. Mice were treated as indicated (Fig. 7) by i.p. injection with either PBS (control) or 1,ug of LPS 1 day before an i.p. injection of 2.5 x 103 live L. monocytogenes cells. Mice receiving no injection of L. monocytogenes served as a control. After 4 weeks, the PECs were collected and tested for their ability to produce lymphokine in the presence of heat-killed L. monocytogenes or SLP. The results from such an experiment are depicted in Fig. 7. Because PECs from mice injected with LPS 1 day before challenge with L. monocytogenes produced no more lymphokine upon incubation with SLP or heat-killed L. monocytogenes than did PECs from nonimmune mice, it appears that LPS does inhibit development of T-cell reactivity to L. monocytogenes. DISCUSSION We have found that macrophages derived from PECs of mice injected i.p. with 1,ug of LPS 3 days before harvest displayed greatly enhanced catabolic activity compared with that of macrophages from untreated mice. This result was not entirely unexpected, for LPS has been shown by many investigators to influence the activation state of macrophages (13). Cohn and Benson (3) demonstrated that injection of microgram quantities of LPS into the peritoneal cavity of mice quickly (4 days) leads to the accumulation of a population of mononuclear phagocytes, mostly macrophages, that express increased amounts of at least three enzymes (acid phosphatase,,b-glucuronidase, and cathepsin). More recently, it has been shown that LPS-elicited peritoneal macrophages, when stimulated with phorbol myristate acetate or phagocytosis, release greater amounts of superoxide anion (O2f) than do normal resident macrophages (4, 11) and that such macrophages are better able to kill pathogenic organisms in vitro (4, 18). Thus, an early response to LPS appears to be the genesis of macrophages that possess increased enzymatic and antimicrobial activities. LPS does not induce activated macrophages after i.p. injection into C3H/HeJ mice (C. Bianco and P. J. Edelson, Fed. Proc., 36:1263, 1977). This abnormal response is due to a single mutation expressed as a cellular defect in response to endotoxin (22). We have demonstrated that the observed LPS-induced increase in catabolic activity is under control of the Lps gene by showing that no increase in catabolic activity was exhibited by LPS-elicited macrophages from C3HeB/HeJ mice (Fig. 3). Many of the functions associated with macrophage activation have been shown to be exhibited upon stimulation of macrophages with LPS in vitro. The addition of as little as 0.1 jxg of LPS per ml to monocytes in vitro leads to changes in cell shape, cytoplasmic organization, number of dense granules, extent of ruffling of the plasma membrane, and enhanced ability of these cells to phagocytize bacteria (2; W. Doe, D. Tang, and P. Henson, Fed. Proc. 36:1263, 1977). Other functions associated with macrophage activation, INFECT. IMMUN. however, are not expressed upon in vitro treatment of macrophages directly with LPS. Wilton et al. (23) found that macrophages incorporate glucosamine only when treated with LPS in the presence of B cells, suggesting that LPS does not directly activate macrophages for glucosamine incorporation but stimulates B lymphocytes, which in turn activate the macrophages. We were not able to stimulate increased catabolic activity upon treatment of macrophages directly with LPS in vitro (data not shown). The cellular requirements for the induction of increased catabolic activity in macrophages by LPS remain to be determined. Because macrophages must take up particulate antigens, catabolize them, and express the antigenic determinants in an energy-requiring process before they can effectively activiate Ia-restricted, antigen-specific T cells (6, 27, 28), it was of interest to determine whether the activation status of macrophages is important in terms of antigen presentation. Using an in vitro system to measure processing and presentation, we determined whether the highly catabolic macrophages that were elicited 3 days after injection of LPS were able to process and present antigen to T cells as efficiently as resident macrophages could. We found that LPS-elicited macrophages processed heat-killed L. monocytogenes slowly (Fig. 4). If fixed within 3 h after the addition of antigen, these cells could not stimulate T-cell activation. Resident macrophages, however, were able to stimulate lymphokine production when fixed only 1.5 h after the addition of antigen. It is possible that these macrophages cannot process antigen effectively during the first few hours after exposure to antigen because the intracellular handling of antigen by these cells is differentially regulated when compared with that by resident macrophages. Activated macrophages have been shown to acquire increased numbers of lysosomes and their associated enzymes (20). Enhanced trafficking of internalized antigen to catabolic compartments within the LPSelicited macrophages may therefore result in extensive antigen degradation. Perhaps these cells are specialized for the elimination of antigen and do not produce the appropriate antigen fragments (processed antigen) needed for stimulation of T cells and the elicitation of specific immune responses. The results presented in Fig. 5 are consistent with this possibility. LPS-elicited macrophages are shown to degrade heat-killed L. monocytogenes to a nonantigenic form at a much faster rate than resident macrophages. Another reason that LPS-elicited macrophages may process and present antigen slowly might be a deficiency of the necessary restriction elements, the Ia molecules. It is apparent from the data presented in Fig. 6 that macrophages elicited 3 days after injection of LPS showed less Ia expression when compared with that of resident or L. monocytogenes-elicited macrophages. LPS-elicited macrophages left unfixed may be able to stimulate T cells because of a decrease in the rate of catabolism during the culture period (Fig. 1), an increase in Ia expression when cultured with T cells and antigen, or both factors. Interestingly, we have found that macrophages from mice injected 7 days before with LPS express high levels of Ia molecules, are not as highly catabolic as macrophages elicited 3 days after injection of LPS, and are much faster at processing antigen than are LPS-elicited macrophages at day 3 (data not shown). Perhaps these observations illustrate the possibility that early in a gram-negative infection, resistance relies primarily on highly catabolic macrophages that are able to kill and degrade the invading bacteria quickly. The

8 VOL. 55, 1987 slower-responding, acquired immune response may be of secondary importance, required to ensure complete clearance of the organisms and to establish immunological memory. These observations may also reflect a necessary compromise that exists between the activation status of the macrophage and the ability of the cell to stimulate specific immune responses through presentation of antigen to T cells. Inducer T-cell clones which corecognize antigen and I region products release macrophage-activating factor shortly after stimulation and can trigger a subset of APCs to express cytolytic activity. This sequence of events plays a central role in the resistance to parasites, bacteria, viruses, and tumor cells. However, Rao et al. (16) have shown that once the macrophages develop cytolytic activity, they do not discriminate between cells in close proximity and will lyse the antigen specific inducer T cells, the very cells responsible for their activated state. Thus, cytocidal activity and antigenpresenting ability may be mutually exclusive to prevent macrophages (activated to kill bacteria or tumor cells) from killing the interacting T cells during the presentation of antigen. Taken together, the results and discussion thus far suggest that LPS, if introduced before antigenic challenge, produces negative regulatory effects on the development of acquired immunity. This effect has been demonstrated. Intravenous injection of a small dose of LPS (1,ug) 24 h before infection with gram-negative (14) or -positive (8, 15) (such as L. monocytogenes) bacteria causes enhanced primary resistance to infection. However, LPS-treated mice in these studies exhibited inferior acquired immunity, as measured by adoptive transfer of immunity to normal mice, delayedtype hypersensitivity to listerial antigens, and uptake of tritiated thymidine by lymphocytes in the spleen (8). We have confirmed these effects on acquired immunity (Fig. 7) by showing that LPS administered 1 day before infection with L. monocytogenes inhibits the development of T-cell reactivity to this bacterium as measured by lymphokine production in response to heat-killed L. monocytogenes or SLP in vitro. Thus, the rapid inflammatory response to LPS includes the development of macrophages which are specialized for antigen degradation functions, with a corresponding deficit in antigen presentation function resulting in decreased acquired immunity. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grants R01 A120215, T32 A (to C.W.C. a recipient of a predoctoral fellowship for research and training in infection and immunity [program director, J. K. Spitznagel]), and K04 A (to H.K.Z., a recipient of a research career development award) from the National Institutes of Health. LITERATURE CITED 1. Allen, P. M., D. I. Belier, J. Braun, and E. R. Unanue The handling of Listeria monocytogenes by macrophages: the search for an immunogenic molecule in antigen presentation. J. Immunol. 132: Bennett, W. E., and Z. A. Cohn The isolation and selected properties of blood monocytes. J. Exp. Med. 123: Cohn, Z. A., and B. Benson The differentiation of mononuclear phagocytes: morphology, cytochemistry, and biochemistry. J. Exp. Med. 121: Cummings, N. P., M. J. Pabst, and R. B. Johnston, Jr Activation of macrophages for enhanced release of superoxide anion and greater killing of Candida albicans by injection of muramyl dipeptide. J. Exp. Med. 152: LPS-ELICITED MACROPHAGES Farr, A. G., J.-M. Kiely, and E. R. Unanue Macrophage- T cell interactions involving Listeria monocytogenes-role of the H-2 gene complex. J. Immunol. 122: Farr, A. G., W. J. Wechter, J.-M. Kiely, and E. R. Unanue Induction of cytocidal macrophages following in vitro interactions between Listeria-immune T cells and macrophages-role of H-2. J. Immunol. 122: Galanos, C., 0. Luderitz, and 0. Westphal A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9: Galleli, A., Y. Le Garrec, and L. Chedid Increased resistance and depressed delayed-type hypersensitivity to Listeria monocytogenes induced by pretreatment with lipopolysaccharide. Infect. Immun. 31: Greenwood, F. C., W. H. Hunter, and J. S. Glover The preparation of I3'l-labeled human growth hormone of highly specific radioactivity. Biochemistry 89: Hodes, R. J., G. B. Ahmann, K. S. Hathcock, H. B. Dickler, and A. Singer Cellular and genetic control of antibody responses in vitro. IV. Expression of Ia antigens on accessory cells is required for response to soluble antigens, including a response under Ir gene control. J. Immunol. 121: Johnston, R. B., Jr., C. A. Godznik, and Z. A. Cohn Increased superoxide anion production by immunologically activiated and chemically elicited macrophages. J. Exp. Med. 148: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Morrison, D. C., and R. J. Ulevitch The effects of bacterial endotoxins on host mediation systems. Bacterial Endotoxins 93: Parent, M. A., F. Boyer, and L. A. Chedid Augmentation de la rdsistance aux infections consecutive a une injection d'endotoxine: mise en evidence du mecanisme par l'association de sulfamide. C.R. Acad. Sci. Ser. D. 260: Parent, M. A., F. J. Parent, and L. A. Chedid Enhancement of resistance to infections by endotoxin-induced serum factor from Mycobacterium bovis BCG-infected mice. Infect. Immun. 28: Rao, A., S. J. Faas, L. J. Miller, P. S. Riback, and H. Cantor Lysis of inducer T cell clones by activiated macrophages and macrophage-like cell lines. J. Exp. Med. 158: Rosenthal, A. S., and E. M. Shevach Function of macrophage in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J. Exp. Med.138: Sasada, M., and R. B. Johnston, Jr Macrophage microbicidal activity: correlation between phagocytosis-associated oxidative metabolism and the killing of Candida by macrophages. J. Exp. Med. 152: Silverstein, S. C., R. M. Steinman, and Z. A. Cohn Endocytosis. Annu. Rev. Biochem. 46: Steinman, R. M., and Z. A. Cohn The metabolism and physiology of the mononuclear phagocytes, p In B. W. Zweifach, L. Grant, and R. T. McCluskey (ed.), The inflammatory process, 2nd ed, vol. 1. Academic Press, Inc., New York. 21. Waravdekar, V., and L. Saslaw A sensitive colorimetric method for the estimation of 2-deoxy sugars with the use of malonaldehyde-thiobarbituric acid reactions. J. Biol. Chem. 234: Watson, J., and R. Riblet Genetic control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that influences mitogenic and immunogenic responses to lipopolysaccharides. J. Exp. Med. 140: Wilton, J., D. Rosenstreich, and J. Oppenheim Activation of guinea pig macrophages by bacterial lipopolysaccharide requires bone marrow derived lymphocytes. J. Immunol. 114: Ziegler, H. K., and C. A. Orlin Analysis of Listeria monocytogenes antigens with monoclonal antibodies. Clin. Invest. Med. 7:

9 1354 CLUFF AND ZIEGLER 25. Ziegler, H. K., L. K. Staffileno, and P. Wentworth Modulation of macrophage Ia-expression by lipopolysaccharide. I. Induction of Ia expression in vivo. J. Immunol. 133: Ziegler, H. K., and E. R. Unanue The specific binding of Listeria monocytogenes-immune T lympocytes to macrophages. I. Quantitation and role of H-2 gene products. J. Exp. Med. 150: INFECT. IMMUN. 27. Ziegler, H. K., and E. R. Unanue Identification of a macrophage antigen-processing event required for I regionrestricted antigen presentation to T lymphocytes. J. Immunol. 127: Ziegler, H. K., and E. R. Unanue Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc. Natl. Acad. Sci. USA 79: