Salmonellae Activate Tumor Necrosis Factor Alpha Production in a ...

INFECTION AND IMMUNITY, Nov. 1997, p. 4624–4633 0019-9567/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 65, No. 11

Salmonellae Activate Tumor Necrosis Factor Alpha Production in a Human Promonocytic Cell Line via a Released Polypeptide FEDERICA CIACCI-WOOLWINE, LOUIS S. KUCERA, STEPHEN H. RICHARDSON, NATHAN P. IYER, AND STEVEN B. MIZEL* Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157 Received 11 June 1997/Returned for modification 11 August 1997/Accepted 26 August 1997

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-a), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-a is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-a production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-a expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-a production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-a levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-a production. Furthermore, the ability to produce the TNF-a inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-a-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-a production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa. TNF-a (3), a product of activated macrophages involved in mediating host defense mechanisms against facultative intracellular pathogens, as well as the pathogenesis of cachexia and septic shock caused by bacterial LPS (7). In the murine model, administration of TNF-a can enhance resistance of the host to infection (37, 39, 40). Although LPS is considered the most potent inducer of TNF-a, purified Salmonella porins have also been shown to trigger activation and cytokine production in human monocytes (16, 17, 32). In primary monocytes and promonocytic cell lines infected with HIV, TNF-a can promote virus production either by itself or in conjunction with other cytokines (8, 12, 14, 15, 20, 25, 33, 43). TNF-a may also be involved in the stimulatory effect of cytomegalovirus (42) or Mycobacterium tuberculosis (29) on HIV replication in peripheral blood mononuclear cells and monocytoid cell lines. In the present study, we investigated the relationship between Salmonella-induced HIV production and TNF-a production, and we also characterized the mechanism by which Salmonella induces TNF-a production.

Salmonellae are gram-negative organisms that are responsible for causing gastroenteritis and enteric fever in humans. Recent studies have shown that Salmonella species can stimulate human immunodeficiency virus (HIV) production in the human promonocytic cell line U1, which is latently infected with the virus (1, 36). Activated macrophages are very susceptible to HIV infection (30), so cell lines such as U1 cells can be used as a model system to study HIV expression and replication and their relationship to macrophage activation. The stimulation of HIV replication in U1 cells is Salmonella specific and is not due to lipopolysaccharide (LPS), since Escherichia coli, Yersinia enterocolitica, and Legionella pneumophila all fail to induce high levels of HIV expression in U1 cells. However, Salmonella strains that are defective in either invasion or survival within macrophages show a decreased ability to stimulate HIV production (36). Andreana et al. (1) reported that antibodies against tumor necrosis factor alpha (TNF-a) reduce the stimulatory effect of the bacteria on HIV production by as much as 50 to 70%. However, the issue of whether TNF-a is the sole factor mediating the effect of the bacteria on viral replication was not addressed. During infection of the gastrointestinal tract, Salmonella can induce an array of proinflammatory cytokines in both epithelial cells and macrophages, including interleukin 1 (IL-1), IL-6, IL-8, TNF-a, and gamma interferon (10, 11, 23, 24, 26, 46). The early pathology observed during Salmonella infection of ligated intestinal loops has been linked to the induction of

MATERIALS AND METHODS Cells. The cell line U1 is a subclone of the human promonocytic cell line U937 that is latently infected with HIV (15). U1 cells were grown in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS) and 50 mg of gentamicin per ml at 37°C in a 5% CO2 atmosphere. The U38 cell line is an HIV-free subclone of U937 cells that is stably transfected with an HIV long terminal repeat-chloramphenicol acetyltransferase reporter construct (13). U38 cells were routinely grown in RPMI 1640 medium supplemented with 10% FBS and 50 mg of gentamicin per ml (complete RPMI medium) at 37°C in a 5% CO2 atmosphere. Both U1 and U38 cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, Md.). Peripheral blood adherent cells (PBAC) were isolated from healthy human donors. Heparinized whole blood was layered over isolymph at a 3:2 ratio

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Wake Forest University Medical Center, Medical Center Blvd., Winston-Salem, NC 27157. Phone: (910) 716-4471. Fax: (910) 716-9928. 4624

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TNF-a INDUCTION BY SALMONELLA

TABLE 1. Bacterial strains used in this study Species or strain

S. enteritidis CDC5 CD5 SM5 S. typhimurium SR11 SB111 SL1344 SJW1103 S. minnesota S. choleraesuis E. coli MM294 Y. enterocolitica 8081c

Parent

CDC5 CDC5

SR11

K-12 8081v

Relevant genotype or characteristic

References

Wild-type Streptomycin resistance pagC::TnphoA

47 47 35

Wild-type invA::Tn5 Wild-type Wild-type Wild-type Wild-type

41 41 21 49 This study This study 34 45

Plasmid cured

and allowed to stand for 1 h. The buffy coat (top layer) was removed and layered again over isolymph at a 4:3 ratio. The tube was centrifuged at 1,100 3 g for 30 min at room temperature, and the top layer containing mononuclear cells was diluted in phosphate-buffered saline (PBS) (pH 7.2). The cells were pelleted by centrifugation, and the cell pellet was resuspended in serum-free RPMI medium. The PBAC were allowed to adhere to tissue culture plates for 2 h at 37°C in 5% CO2. The plates were washed to remove nonadherent cells. The adherent cells were incubated in complete RPMI medium overnight at 37°C in 5% CO2. In order to induce LPS tolerance, PBAC were incubated overnight in complete RPMI medium containing 1 mg of Salmonella LPS (Sigma, St. Louis, Mo.) per ml. Bacteria. All of the wild-type and mutant bacterial strains used in this study are listed in Table 1. Strains CDC5str (CD5), SM5T (SM5), and 8081c were provided by Virginia Miller; the SB111 mutant was provided by Jorge Gala`n; MM294 was provided by Ian Blomfield; SL1344 was provided by Charles J. Dorman, and SJW1103 was provided by Robert Macnab. Stocks of all bacterial strains were stored at 270°C in 25% (vol/vol) glycerol. Bacteria were grown either in LuriaBertani broth (LB) or in RPMI 1640 medium (without serum). LB cultures were grown overnight at 37°C on a roller drum, whereas RPMI 1640 cultures were grown at 37°C overnight without aeration to simulate growth conditions during infection experiments. Yersinia cultures were grown in LB at 30°C on a reciprocal shaker. Bacterial concentrations were determined turbidimetrically (600 nm). ELISA for p24 antigen. The amount of p24gag antigen was used as a measure of virus production by U1 cells. The culture medium from U1 cells was assayed at 1:50 to 1:1,000 dilutions with a commercial p24 enzyme-linked immunosorbent assay (ELISA) kit (Organon Teknika, Durham, N.C.). ELISA for TNF-a. The total amount of TNF-a produced (cell associated and released) was measured with a commercial ELISA kit (Cistron, Pine Brook, N.J.). Cultures of U1 and U38 cells were treated with Triton X-100 (TX-100) to a final concentration of 0.5%. Undiluted 100-ml aliquots of the TX-100 lysates were tested in the assay according to the manufacturer’s protocol. Bacterial infection of U1 cells. Prior to infection, 1 3 106 to 3 3 106 bacteria from LB cultures aerated overnight were centrifuged at 5,000 3 g for 5 min, washed twice in PBS, and resuspended in RPMI 1640 medium without supplements. U1 cells (1 3 106 to 3 3 106) were infected with each of the bacterial strains at a multiplicity of infection (MOI) of 1, either in the presence or absence of anti-TNF-a monoclonal antibodies (final dilution, 1:100; Genzyme, Cambridge, Mass.), for 2 h at 37°C. Cells were washed to remove unbound bacteria and resuspended in RPMI 1640 medium containing 20% FBS and gentamicin with or without anti-TNF-a antibodies. After 72 h, the cells were removed by centrifugation, the culture medium was adjusted to 0.5% TX-100 and assayed in the p24 ELISA at dilutions of 1:50 to 1:1,000. Bacterial infection of U38 cells and PBAC. Prior to infection, an appropriate number of bacteria were centrifuged, washed as described above, and resuspended in gentamicin-free RPMI 1640 medium lacking FBS. They were then added to 3 3 106 to 1 3 107 U38 cells or PBAC in a 24-well tissue culture plate at an MOI of 10. After a 2-h incubation at 37°C, U38 cells or PBAC were washed and incubated in gentamicin-containing RPMI 1640 medium to kill extracellular bacteria. After an additional 2 h, the cultures were lysed in 0.5% TX-100 and the extracts were used in the TNF-a ELISA. Preparation of Salmonella CD5 conditioned medium (CM). Cultures of Salmonella grown overnight were centrifuged at 9,000 3 g for 10 min to pellet bacteria, and the CM was sterilized by passage through a 0.2-mm-pore-size Millipore filter. CM samples were stored at 4°C. The CM samples were added to U38 cells or PBAC at the dilutions indicated in the figure legends in a total volume of 0.5 to 1 ml. All incubations were carried out in tissue culture plates at 37°C in 5% CO2 for 4 h (preliminary experiments revealed that TNF-a production was maximal by 4 h).

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Trypsin treatment of CD5 CM samples. The CM from cultures of S. enteritidis CD5 grown overnight were filter sterilized and incubated either alone or with trypsin (at a final trypsin concentration of 10 mg/ml) at 37°C for 10 min. Following this step, three protease inhibitors were added to the trypsin-containing samples at the following concentrations: antipain, 100 mM; leupeptin, 100 mM; and aprotinin, 2 mg/ml. The samples containing inactive trypsin were prepared by mixing CD5 CM with trypsin that had previously been incubated with the same three inhibitors at 37°C for 10 min. After the addition of the inhibitors, the samples were incubated on ice for an additional 10 min before being added to the U38 cells at the dilutions indicated in the figure legends. Biochemical analysis of Salmonella CM. To assess the sensitivity of the TNF-a inducer to temperature, CM from cultures of CD5 cells grown overnight were filter sterilized and incubated for 10 min at either 100, 37, or 4°C. Samples were then centrifuged at 23,000 3 g for 5 min, and the supernatants were used to stimulate U38 cells for 4 h at the dilutions indicated in the figure legends. The pH sensitivity of the TNF-a inducer was evaluated by the addition of 2.5 M glycine (pH 3 or 6.4) to filter-sterilized CD5 CM to a final concentration of 50 mM and pH 3 or 6.8. The samples were incubated on ice for 30 min and then dialyzed overnight against LB at 4°C with 10,000-molecular-weight-cutoff (MWCO) dialysis cassettes (Pierce, Rockford, Ill.). Adding 2.5 M glycine at pH 6.4 to CM did not alter its initial pH (6.8) but provided appropriate control conditions for the presence of glycine and for sample dilution and loss caused by the dialysis process. Each sample was used on U38 cells as described above. For the urea treatment, 1.8 g of urea was dissolved into 2 ml of CD5 CM to obtain an 8 M solution. Control CM was diluted with LB to the same final volume (3 ml) and dialyzed with the urea-treated CM at room temperature twice for 3 h and then overnight at 4°C against LB. Dialysis was performed with 10,000-MWCO dialysis cassettes (Pierce). The recovered samples were used as stimuli on U38 cells as described above. Gel filtration chromatography of Salmonella CM. Salmonella from a culture grown overnight were resuspended in RPMI 1640 medium containing 0.01% bovine serum albumin (BSA) and grown at 37°C for 2 h without agitation. The CM were collected and filter sterilized before being concentrated 100-fold by ultrafiltration through a YM10 membrane (10,000 MWCO; Amicon). An aliquot of the concentrate (1.3 ml or 1% of the column bed volume) was loaded onto a G200 Sephadex column equilibrated with 150 mM NaCl and 50 mM Na2HPO4, pH 7.2, and the column was run at a rate of 12 ml/h. Two-milliliter fractions were collected and refrigerated until analyzed for the presence of TNF-a inducer activity. Blue dextran was used to determine the void volume of the column, and aldolase, BSA, and cytochrome c were used for molecular mass calibration. Each column fraction was tested for TNF-a-inducing activity on U38 cells at a 1:100 dilution.

RESULTS Anti-TNF-a antibodies inhibit Salmonella-induced HIV replication. In view of the well-documented ability of IL-1 (8, 19, 44) and TNF-a (8, 12, 14, 20, 25, 33) to promote HIV replication, we tested the effect of neutralizing antibodies against these two cytokines on Salmonella-induced HIV replication in U1 cells. Although anti-IL-1b antibodies had no effect (data not shown), anti-TNF-a antibodies significantly reduced the response to Salmonella (Table 2). When U1 cells were incubated with wild-type S. enteritidis CD5 in the presence of antiTNF-a antibodies, the extent of HIV replication was decreased by 50 to 80% in three independent experiments. Consistent with previous results (36), mutant S. enteritidis SM5 and S. typhimurium SB111 strains were much less effective inducers of HIV production than the wild-type S. enteritidis CD5 (36). Nonetheless, anti-TNF-a antibodies also reduced the levels of HIV replication in response to these mutant strains. In addition, neither E. coli nor Y. enterocolitica stimulated significant virus replication. The observed inhibition of HIV replication by anti-TNF-a antibodies is consistent with the hypothesis that TNF-a plays a critical role in Salmonella-induced HIV replication. Effects of various Salmonella strains on TNF-a expression. In order to determine the extent of TNF-a involvement in Salmonella-induced macrophage activation and HIV replication, we investigated the relationship between the ability of a Salmonella strain to induce TNF-a and HIV replication. The wild-type S. enteritidis CD5 strain induced substantial levels of both TNF-a and HIV replication in U1 cells (Tables 2 and 3). However, in marked contrast to their limited ability to promote

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TABLE 2. Anti-TNF-a antibodies block Salmonella-induced HIV replication in U1 cellsa p24 level (pg/ml) Bacterium

% Inhibition by anti-TNF-a

Without anti-TNF-a

With anti-TNF-a

23 13

4 1

83 92

S. typhimurium SB111

2

0

100

Y. enterocolitica 8081c

0

0

E. coli MM294

0

0

Control

0

0

S. enteritidis CD5 SM5

6

a

U1 cells (10 ) were infected with bacteria from cultures grown overnight at an MOI of 1 for 2 h either in the presence or absence of anti-TNF-a antibodies. U1 cells were then washed and transferred to gentamicin-containing RPMI to kill extracellular bacteria. Anti-TNF-a antibodies were added to the U1 cells that had been incubated with the antibody during the initial 2 h. Cells were removed by centrifugation after 72 h, and the culture medium was adjusted to 0.5% TX-100 prior to assay with a p24 ELISA kit. Values between 50 and 80% inhibition of CD5 were obtained in three separate experiments.

HIV replication (36) (Table 2), the S. enteritidis SM5 and S. typhimurium SB111 strains in general induced significant levels of TNF-a (Table 3). Although the levels of induction obtained with SM5 were comparable to those observed with CD5, we observed some variability when the SB111 strain was used as the stimulus. Prompted by our results on Salmonella-induced TNF-a synthesis in U1 cells, we continued our experiments with the non-HIV-infected U38 cells, which are closely related to U1 cells. U38 cells were incubated for 2 h with Salmonella CD5, SM5, or SB111 at a bacterial MOI of 10. The U38 cells were then washed and incubated for an additional 2 h in gentamicin-containing medium to kill the extracellular bacteria. Subsequently, the U38 cells were harvested and lysed, and the total amount of TNF-a (cell associated and released) was determined by ELISA. E. coli MM294 was included to control for nonspecific cell activation. Y. enterocolitica 8081c served as a control for the effect of an intracellular pathogen on macrophage activation. This strain was chosen because it has been cured of the virulence plasmid encoding YopB, a protein which has been shown to downregulate TNF-a expression in primary monocytes (6). The results of three independent experiments are shown in Fig. 1. As observed in U1 cells, all three Salmonella strains stimulated significant levels of TNF-a production in U38 cells. Conversely, neither E. coli nor Y. enterocolitica had a similar effect on cytokine production. Our results with

FIG. 1. Effects of various gram-negative bacteria on TNF-a production on U38 cells. U38 cells (106) were incubated for 2 h with bacteria at an MOI of 10 in 1 ml of RPMI 1640 medium. The U38 cells were then washed and resuspended in RPMI 1640 medium containing gentamicin (50 mg/ml) and 10% FBS for an additional 2 h. At this time, TX-100 was added to each sample at a final concentration of 0.5% to lyse the cells and the lysates were assayed by ELISA. Control values have been subtracted for each experiment. The control values for the experiments were as follows: experiment 1, 0.6 pg/106 cells; experiment 2, 7 pg/106 cells; experiment 3, 17 pg/106 cells.

U1 and U38 cells confirm and extend previous observations that Salmonella has the ability to upregulate TNF-a expression in cells of the promonocytic lineage (1). However, our findings demonstrate that although TNF-a is required for Salmonellainduced HIV replication, there must be an additional signal

TABLE 3. Wild-type and mutant Salmonella strains induce TNF-a in HIV-infected U1 cellsa Stimulus

TNF-a level (pg/106 cells)

CD5............................................................................................ 160 6 35 SM5............................................................................................ 146 6 27 SB111......................................................................................... 66 6 35 None .......................................................................................... 2 6 3 a U1 cells (106) were incubated with bacteria at an MOI of 1 for 2 h in gentamicin- and FBS-free RPMI 1640 medium. The cells were then washed and resuspended in gentamicin-containing RPMI 1640 medium for an additional 2 h. At this time U1 cells were lysed by the addition of 1% TX-100 and the total amount of TNF-a in the lysates was measured by ELISA. Values shown are the means and standard deviations of three independent experiments.

FIG. 2. Salmonella CM induces TNF-a expression in U38 cells. Serial dilutions of filtered CM from cultures of S. enteritidis CD5 grown overnight in LB (■) or RPMI 1640 medium (Œ) were tested on U38 cells. After a 4-h incubation, cells were harvested and assayed as described in Materials and Methods. Unstimulated cells produced no detectable TNF-a.

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FIG. 3. Time course of the accumulation of TNF-a in U38 cells in response to live Salmonella and Salmonella CM. (a) S. enteritidis CD5 bacteria were incubated with U38 cells at an MOI of 10 in RPMI. Cells were harvested at the indicated times, and the total amount of TNF-a was measured by ELISA. The background control values for these experiments were 2 and 7 pg/106 cells. (b) U38 cells were incubated with filter-sterilized CD5 CM at a 1:50 dilution. The U38 cells were lysed by the addition of TX-100 at the indicated times. The background value for the experiments were 4, 4, and 5 pg/106 cells.

that is optimally delivered by Salmonella strains that can invade and survive. Furthermore, the ability to induce high-level TNF-a production is not a general effect of gram-negative or intracellular pathogens, since neither E. coli nor Y. enterocolitica induced significant TNF-a production in U38 cells. Salmonella CM activates TNF-a expression in U38 cells. Several organisms, such as Mycoplasma hyorhinis, Propionibacterium acnes, and M. tuberculosis (5, 28, 48), have been shown to upregulate TNF-a production in monocytes and macrophages. Interestingly, they all appear to do so through released products. In order to determine if Salmonella-induced TNF-a synthesis is also mediated by a released product, we tested the ability of CM from cultures of S. enteritidis CD5 to induce TNF-a synthesis in cultures of U38 cells. The bacteria were grown overnight in LB or in RPMI 1640 medium, and the CM were filter sterilized and added to cultures of U38 cells. The amount of TNF-a in these cells was determined by ELISA. Since the bacteria are routinely grown in LB but are washed and resuspended in RPMI 1640 medium just prior to being added to the cells, we tested the CM from cells grown in each of these media. The results of a representative experiment with LB and RPMI 1640 CM are presented in Fig. 2. The CM from cultures of the CD5 strain stimulated the production of TNF-a in a concentration-dependent manner; 50% of the maximal stimulation was achieved with LB CM at a dilution of 1:100. Although the CM from CD5 bacteria grown in RPMI 1640 medium also induced TNF-a synthesis in U38 cells, it was less active than the LB CM, with 50% of the maximal response achieved with a 1:50 dilution as opposed to a 1:100 dilution. Based on these results, we defined 1 unit of activity as the dilution that induces 50% of the maximal level of TNF-a. Kinetics of TNF-a production in response to live Salmonella and to bacterial CM. In order to determine the relative contribution of the released product to the overall TNF-a-inducing effect of the bacteria, we studied the kinetics of cytokine induction in response to Salmonella and to their CM. In the first set of experiments, U38 cells were incubated with S. enteritidis CD5 and harvested every 30 min. In a second set of experiments, U38 cells were incubated with Salmonella CM at

a 1:50 dilution and harvested at the same times. The amount of TNF-a produced by U38 cells was measured by ELISA. The results of these experiments are shown in Fig. 3. The data presented show that TNF-a production in U38 cells is markedly elevated within 60 min after the addition of either Salmonella (Fig. 3a) or its CM (Fig. 3b). Because the bacteria are thoroughly washed before being added to the U38 cells, these results suggest that the TNF-a inducer is constitutively released by the bacteria. Indeed, Salmonella CM contain significant levels of TNF-a-inducing activity after only 1 min of bacterial growth in culture (data not shown). The observation that the levels and the kinetics of TNF-a production are similar in U38 cells stimulated with bacteria (Fig. 3a) or with CM (Fig. 3b) is consistent with the conclusion that the released product is an important Salmonella-derived signal for TNF-a synthesis. The ability to induce TNFa is not serotype specific. There are at least 60 Salmonella serotypes that can be distinguished biochemically. In order to determine if the ability to induce TNF-a is conserved among these variants, we tested CM from five wild-type strains representative of different serotypes on U38 cells and measured TNF-a levels in the cell cultures. The results of these experiments are shown in Fig. 4. All of the Salmonella serotypes were able to stimulate TNF-a to significant levels in U38 cells. These results demonstrate that the ability to induce TNF-a is not unique to only a single Salmonella serotype but is a shared activity of several different serotypes. The TNF-a-inducing factor(s) is a trypsin-sensitive polypeptide. The results presented so far are consistent with the idea that Salmonella activates TNF-a synthesis in U38 cells through a factor that is released under normal bacterial growth conditions. U38 cells respond quickly to this stimulus. In order to determine if the TNF-a inducer is a polypeptide, the Salmonella CM was treated with trypsin prior to being assayed for TNF-a-inducing activity. U38 cells were incubated with untreated Salmonella CM or with CM treated with 10 mg of

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FIG. 4. TNF-a induction in U38 cells by CM from different Salmonella serotypes. CM from five Salmonella strains were used to stimulate U38 cells at a final dilution of 1:160 as described in Materials and Methods. Unstimulated cells expressed no detectable levels of TNF-a.

trypsin per ml for 10 min (the trypsin was subsequently inactivated with a proteinase inhibitor mix of leupeptin and antipain at final concentrations of 100 mM and aprotinin at a final concentration of 2 mg/ml) or with CM treated with inactive trypsin (trypsin treated with the inhibitors for 10 min prior to being mixed with the CM). All of the samples were tested on U38 cells at three different dilutions. U38 cells were harvested 4 h after addition of the stimulus, and total TNF-a was measured by ELISA. The means and standard deviations from four separate experiments are shown in Fig. 5. Trypsin treatment markedly reduced the TNF-a-inducing activity present in the Salmonella CM. The observation that CM treated with inactive trypsin was as active as the untreated samples demonstrates that the observed loss of activity is not due to a carryover effect of trypsin on the U38 cells. Similar results were obtained with RPMI 1640 CM (data not shown). Taken together, these data indicate that the TNF-a inducer factor present in Salmonella CM is a trypsin-sensitive polypeptide. Biochemical characterization of the TNF-a inducer. In order to further characterize the TNF-a-inducing activity contained in Salmonella CM, we tested its sensitivity to temperature, urea denaturation, and low pH. The results of these experiments are shown in Fig. 6. Heating active CM to 100°C for 10 min did not result in a decrease in its ability to induce TNF-a in U38 cells (Fig. 6a). Likewise, lowering the pH of active CM to 3 (Fig. 6b) or reversibly denaturating the activity with 8 M urea (Fig. 6c) did not result in decreased TNF-a induction. Thus, it appears that the TNF-a inducer is quite stable. Gel filtration chromatography of the Salmonella-derived TNF-a-inducing activity. In order to determine the molecular mass of the TNF-a inducer released by Salmonella, bacteria were grown in RPMI 1640 medium containing 0.01% BSA (the addition of BSA has been observed to increase the stability of the inducer in this medium) and the sterile CM was concentrated 100-fold by Amicon ultrafiltration. G200 Sephadex was used to fractionate the CM. The fractions between the void

volume and the RPMI phenol red elution volume were tested on U38 cells for TNF-a-inducing activity. A representative elution profile of the TNF-a-inducing activity is presented in Fig. 7. The majority of the activity was recovered in two peaks, one corresponding to an apparent molecular mass of 150 kDa and a second one of approximately 110 kDa. Dilution analysis

FIG. 5. The TNF-a-inducing activity in Salmonella CM is sensitive to trypsin. Active CD5 CM was incubated at 37°C for 10 min with or without 10 mg of trypsin per ml or with inactive trypsin (a mixture of 100 mM antipain, 100 mM leupeptin, and 2 mg of aprotinin per ml was used to inhibit trypsin). The trypsin in the experimental samples was inactivated with the three inhibitors for 10 min on ice before samples were added to the cells. Serial dilutions of the CM were tested on U38 cells as described in Materials and Methods. The means and standard deviations of four separate experiments are shown. Untreated control cells expressed no detectable TNF-a.

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FIG. 6. The TNF-a-inducing activity contained in Salmonella CM is insensitive to temperature, low pH, and 8 M urea. (a) Effects of different temperatures on the stability of Salmonella CM. Samples of active CM were incubated at either 4, 37, or 100°C for 10 min. The samples were then centrifuged and used to stimulate U38 cells as described in Materials and Methods. The unstimulated cell control sample contained 3 6 0.3 pg of TNF-a/106 cells. (b) Effect of low pH on Salmonella CM. Active CM samples were incubated with 50 mM glycine at pH 3 or 6.8 on ice for 30 min. The samples were then dialyzed overnight against LB at 4°C. Serial dilutions of the treated CM were tested on U38 cells for the ability to induce TNF-a. No TNF-a was detected in the unstimulated control samples. (c) Effect of treatment with 8 M urea. Salmonella CM samples were treated with (1) and without (2) 8 M urea and then dialyzed against LB to remove the denaturing agent. The samples were then used to stimulate U38 cells at the indicated dilutions. Background TNF-a production was 3 6 1 pg/106 cells.

revealed that the activities of the two species decreased in parallel, suggesting that they are present in similar amounts (although it is also possible that one species is more potent than the other and can stimulate TNF-a to comparable levels even at lower concentrations). Relative contributions of Salmonella components to TNF-a induction in PBAC. In order to determine the relative contributions of the trypsin-sensitive Salmonella-derived TNF-a inducer and LPS to the overall effect of Salmonella on TNF-a production in human primary monocytes, we tested the effects of Salmonella and E. coli on peripheral blood monocytes with and without prior exposure of the cells to LPS. As previously shown (18), freshly isolated monocytes from volunteers injected with E. coli endotoxin show a significant decrease in the

levels of TNF-a produced in response to LPS stimulation in vitro. We therefore incubated LPS-nonresponsive and LPSresponsive PBAC with S. enteritidis CD5 or E. coli MM294 to determine the relative contributions of the TNF-a inducer and LPS in TNF-a induction. The results of these experiments are shown in Fig. 8. Monocytes that have not been exposed to LPS respond to LPS as well as Salmonella and E. coli with high levels of TNF-a. However, PBAC that have been exposed to LPS and are now tolerant are still capable of producing TNF-a following exposure to Salmonella, whereas they are significantly less responsive to E. coli. These data suggest that a Salmonella-specific stimulus other than LPS can trigger TNF-a accumulation. Different Salmonella serotypes induce TNF-a in peripheral blood monocytes. In order to determine whether different Salmonella serotypes can induce TNF-a in monocytes as in the U38 cell line, we tested CM from five different Salmonella strains on LPS-nonresponsive PBAC. The results of these experiments are shown in Fig. 9. Treatment with LPS overnight made the cells virtually unresponsive to subsequent exposure to LPS. As previously shown for U38 cells (Fig. 4), all of the Salmonella strains tested induced significant levels of TNF-a in PBAC. Since LPS alone had no effect on TNF-a production in the tolerant cells, it is clear that monocytes are also sensitive to the non-LPS TNF-a inducer of Salmonella.

4630


FIG. 7. Gel filtration chromatography of active Salmonella CM. An aliquot (1.3 ml) of concentrated CM from CD5 grown in RPMI 1640 medium for 2 h was chromatographed on a Sephadex G200 column at a flow rate of 12 ml/h. Each fraction between the void volume (V0) and the elution volume of the phenol red indicator dye was assayed on U38 cells at a 1:100 dilution as described in Materials and Methods. The elution volumes of three standards are shown. The results are representative of three separate experiments.

DISCUSSION TNF-a is a product of activated macrophages that plays an important role in the pathogenesis of viral and inflammatory diseases. Our results as well as those of Andreana et al. (1) demonstrate that TNF-a production following Salmonella infection is required for enhanced HIV replication in a monocyte/macrophage-like cell line, U1, which harbors the virus in a latent phase. Anti-TNF-a antibodies dramatically reduced HIV replication in U1 cells infected with Salmonella (Table 1). The addition of neutralizing antibodies significantly inhibited virus production in U1 cells regardless of the strain used, suggesting that although mutant Salmonella strains cannot induce the same levels of HIV as the wild-type strain, in all cases, the effect is mediated in part by TNF-a. These observations prompted us to characterize the mechanism of TNF-a induction by Salmonella in the U38 cell line, also a subclone of U937, like U1 cells. Our results indicate that the ability of Salmonella strains to induce high-level TNF-a expression is not mediated by LPS, as it is not shared by other gram-negative bacteria (Fig. 1). S. typhimurium, S. enteritidis, S. minnesota, and S. choleraesuis were all able to stimulate TNF-a accumulation in U38 cells and PBAC, but, interestingly, neither invasion nor intracellular survival appeared to be necessary for this effect, since mutant strains that are deficient for these two phenotypic characteristics (SB111 and SM5) showed no significant impairment in TNF-a induction in U38 cells compared to the wild-type strain. Taken together, our results are consistent with the conclusion that, although TNF-a synthesis is essential for Salmonellamediated induction of HIV replication, optimal stimulation requires additional signaling that is more efficiently delivered by Salmonella strains that are able to invade and survive intracellularly. This additional signaling may be due to the same factor that induces TNF-a or to a distinct factor that is also derived from the bacteria. If only one factor were responsible for TNF-a production and viral replication, then its ability to promote HIV production must be dependent upon its contin-

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ued expression by viable, internalized bacteria, as would be the case with the wild-type strains of Salmonella. Thus, the lack of a direct correlation between the ability of SM5 and SB111 to induce TNF-a and virus replication would be due to the limited capacity of these strains to deliver the activating signal intracellularly. This hypothesis would provide an explanation for the observation that Salmonella CM is not as effective as live bacteria in inducing HIV replication in U1 cells (unpublished observation). However, it is also possible that a signal that is distinct from the TNF-a inducer is required for optimal HIV production in response to Salmonella. Based on the reduced ability of the SM5 strain to survive within macrophages and to induce HIV production, this putative second signal would be effective only when delivered intracellularly over a period of more than 4 h, the point at which the intracellular survival of the SM5 strain is dramatically reduced, relative to the wild-type CD5 strain (36). Clearly, additional studies are required to differentiate between these two possibilities. As mentioned above, organisms other than Salmonella can activate macrophages to synthesize and release TNF-a via secreted products. Likewise, when Salmonella CM was tested on U38 cells, TNF-a was induced with the same kinetics as when live bacteria were used as the stimulus (Fig. 3). The activity in the CM was titratable and present at concentrations ranging from 50 to 100 U/ml, depending on the type of medium in which the bacteria were grown. We have observed that Salmonella-conditioned LB consistently contains more activity than RPMI 1640 CM. One possible explanation is that the activity undergoes nonspecific loss due to the low protein concentration of this medium or is proteolytically degraded. These possibilities are consistent with the observation that BSA can stabilize the activity present in RPMI 1640 CM. However, we

FIG. 8. Relative contribution of LPS to the overall level of TNF-a induction by Salmonella in cultures of human monocytes. LPS-nonresponsive and LPSresponsive tolerant PBAC from the same donor were infected with either S. enteritidis CD5 or E. coli MM294 at an MOI of 10 for 2 h. The PBAC were then washed, and gentamicin was added to the medium to kill extracellular bacteria. Following an additional 2-h incubation, the cultures were lysed and the amount of TNF-a was determined in each sample. LPS (1 mg/ml) was used as the control for the establishment of tolerance. The TNF-a values shown have been normalized with respect to the unstimulated controls. For the LPS-nonresponsive PBAC, the background value was 9 6 1 pg of TNF-a/106 cells. For the LPSresponsive cells, the value was 0 6 1 pg/106 cells.

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FIG. 9. TNF-a induction in human PBAC by different Salmonella serotypes. CM was used to stimulate LPS-nonresponsive PBAC under the same experimental conditions described for U38 cells. The PBAC were incubated overnight in complete RPMI 1640 medium containing 1 mg of LPS per ml and then washed before stimulation with CM or LPS (also at 1 mg/ml). In the experiment shown, unstimulated cells produced 9 pg of TNF-a/106 cells.

cannot rule out the possibility that a component of LB specifically protects the TNF-a inducer from degradation or stimulates its release by Salmonella. The results of kinetics experiments demonstrate that macrophages respond very rapidly to the stimulus present in Salmonella CM and that the release of the inducer is constitutive (TNF-a levels are increased by 60 min in response to the bacteria as well as the soluble inducer). The observed rise in TNF-a levels could be a result of increased TNF-a gene transcription or mRNA stability. The mechanisms involved in TNF-a upregulation at the molecular level are currently under investigation. The trypsin sensitivity of the TNF-a inducer indicated that it is at least in part a polypeptide. In this regard, purified Salmonella porins have been shown to activate TNF-a in human blood monocytes (16). However, our data suggest that the TNF-a inducer that we have found is not a porin. We have tested an ompR::Tn10 mutant strain of S. typhimurium SL1344, CJD359 (9), which showed no decrease in its ability to upregulate TNF-a expression (unpublished observation). Gel filtration chromatography of the TNF-a inducer revealed two peaks of activity with apparent molecular masses of 150 and 110 kDa. It is possible that these peaks correspond to two distinct products that can act independently, or they may also represent different oligomeric forms of a single protein. Also, we cannot at present rule out the possibility that the TNF-a inducer is actually a lower-molecular-weight protein that nonspecifically interacts with the BSA present in the concentrated CM. In preliminary experiments, we have found that TNF-a production in cultures of human peripheral blood monocytes is induced by the same Sephadex G200 fractions as in the case of U38 cells. The observation that the TNF-a-inducing activity in Salmonella CM is resistant to high temperatures, reversible urea denaturation, and low pH suggests that the trypsin-sensitive protein is relatively stable. In view of the central role that appropriate control of cyto-

kine production plays in the host response to pathogenic bacteria, the abilities of an enteric pathogen to activate macrophages and to induce expression of proinflammatory mediators such as TNF-a are relevant and applicable to more than one disease state. As mentioned above, Salmonella enhances the rate of viral replication in cells chronically infected with HIV, and anti-TNF-a antibodies can block a substantial part of this effect. Our results expand on and partially refine the observation that TNF-a is a major factor in Salmonella activation of HIV production. As confirmed in U1 cells (Table 3), Salmonella invasion and survival mutants that are defective for HIV induction do not show the same defect in TNF-a induction compared to wild-type bacteria. This argues that TNF-a, although important, is not the only mediator in this process. However, when present in sufficiently high concentrations, TNF-a can induce HIV replication in the absence of other stimuli (12, 20, 25, 33). Production of TNF-a appears to mediate some of the early inflammatory and cytopathic responses observed during Salmonella infection of the intestinal tract (3, 4). In particular, anti-TNF-a antibodies administered to mice before challenge with virulent Salmonella strains can have the antithetic effects of increasing resistance to infection (40) and eliminating some of the pathology observed in the intestines (3). Thus, in addition to its relevance to the pathogenesis of AIDS, understanding the mechanism by which a released Salmonella protein stimulates gene expression in monocytes may shed light upon some of the signal transduction pathways involved in macrophage activation. The interaction between macrophages and facultative intracellular pathogens, such as Salmonella, can have a number of deleterious consequences. For example, by activating TNF-a production, Salmonella may indirectly affect the life cycle of coinfecting pathogens as well as damage infected tissues of the host. During Salmonella infection of murine ligated intestinal loops, the levels of IL-1a, IL-6, gamma

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interferon, and TNF-a are elevated (26), and the administration of recombinant TNF-a results in tissue damage similar to that which follows Salmonella infection (3). In addition, evidence is accumulating that Salmonella infection can result in the death of cells of the intestinal epithelium (including M cells) (22, 27) as well as monocytes/macrophages (31). Whether TNF-a is directly responsible for this effect has not been determined, but one study has suggested that IL-10 may inhibit apoptosis of Salmonella-infected macrophages by preventing the accumulation of TNF-a or by stimulating shedding of the TNF-a receptor from these cells (2). During infection, the immune system of the host is exposed and responds to different bacterial components by releasing TNF-a. LPS is an extremely potent stimulus for TNF-a production, but other Salmonella-derived factors may also contribute to cytokine expression (16). At the onset of infection, macrophages, which represent a first line of defense against pathogens such as Salmonella, are most sensitive to LPS. LPS activates these cells to produce and secrete proinflammatory cytokines. However, after prolonged LPS stimulation (as occurs during sepsis), these cells become tolerant to the stimulus (38). At this point, other bacterial components might emerge as stimuli for continued cytokine production. The trypsin-sensitive TNF-a inducer is likely to be such a stimulant. ACKNOWLEDGMENTS We thank Virginia Miller for the CD5 and SM5 Salmonella strains, and for the 8081c Yersinia strain, Jorge Gala`n for the S. typhimurium SB111 strain, Ian Blomfield for the E. coli MM294 strain, Charles Dorman for the SL1344 and CJD359 strains, and Robert Macnab for the SJW1103 strain. Tissue culture reagents and services were provided by the Tissue Culture Core Laboratory of the Comprehensive Cancer Center of Wake Forest University. This project was supported in part by a Venture Grant from Bowman Gray School of Medicine of Wake Forest University and by NIH grant AI 38670. The Tissue Culture Core Laboratory of the Comprehensive Cancer Center of Wake Forest University is supported in part by NIH grant CA 12197. REFERENCES 1. Andreana, A., S. Gollapudi, C. H. Kim, and S. Gupta. 1994. Salmonella typhimurium activates human immunodeficiency virus type 1 in chronically infected promonocytic cells by inducing tumor necrosis factor-a production. Biochem. Biophys. Res. Commun. 201:16–23. 2. Arai, T., K. Hiromatsu, H. Nishimura, Y. Kimura, N. Kobayashi, H. Ishida, Y. Nimura, and Y. Yoshikai. 1995. Endogenous interleukin-10 prevents apoptosis in macrophages during Salmonella infection. Biochem. Biophys. Res. Commun. 213:600–607. 3. Arnold, J. W., G. R. Klimpel, and D. W. Niesel. 1993. Tumor necrosis factor (TNFa) regulates intestinal mucus production during salmonellosis. Cell. Immunol. 151:336–344. 4. Arnold, J. W., D. W. Niesel, C. R. Annable, C. B. Hess, M. Asuncion, Y. J. Cho, J. W. Peterson, and G. R. Klimpel. 1993. Tumor necrosis factor-a mediates the early pathology in Salmonella infection of the gastrointestinal tract. Microb. Pathog. 14:217–227. 5. Averill, L., Z. Toossi, H. Aung, W. H. Broom, and J. J. Ellner. 1995. Regulation of production of tumor necrosis factor alpha in monocytes stimulated by the 30-kilodalton antigen of Mycobacterium tuberculosis. Infect. Immun. 63:3206–3208. 6. Beuscher, H. U., F. Rodel, A. Forsberg, and M. Rollinghoff. 1995. Bacterial evasion of host immune defense: Yersinia enterocolitica encodes a suppressor for tumor necrosis factor alpha expression. Infect. Immun. 63:1270–1277. 7. Beutler, B., and A. Cerami. 1989. The biology of cachectin/TNFa—a primary mediator of the host response. Annu. Rev. Immunol. 7:625–655. 8. Conaldi, P. G., C. Serra, A. Dolei, F. Basolo, V. Falcone, G. Mariani, P. Speziale, and A. Toniolo. 1995. Productive HIV-1 infection in human vascular endothelial cells requires cell proliferation and is stimulated by combined treatment with interleukin-1 beta plus tumor necrosis factor alpha. J. Med. Virol. 47:355–363. 9. Dorman, C. J., S. Chatfield, C. F. Higgins, C. Hayward, and G. Dougan. 1989. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect. Immun. 57:2136–2140.

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