Killed Pseudomonas aeruginosa - Infection and Immunity - American ...

2 downloads 0 Views 1MB Size Report
Nov 1, 1982 - Whole antibiotic-killed classic Pseudomonas aeruginosa organisms elicited ... that a high dose of P. aeruginosa, when added to lymphocyte ...
Vol. 39, No. 2

INFECTION AND IMMUNITY, Feb. 1983, p. 630-637

0019-9567/83/020630-08$02.00/0 Copyright © 1983, American Society for Microbiology

Suppression of In Vitro Lymphocyte DNA Synthesis by Killed Pseudomonas aeruginosa HAYA R. RUBIN,t RICARDO U. SORENSEN,* PATRICIA A. CHASE, AND JEFFREY D. KLINGER Departments of Pediatrics and Pathology, Case Western Reserve University School of Medicine, and Rainbow Babies and Childrens Hospital, Cleveland, Ohio 44106

Received 8 July 1982/Accepted 1 November 1982

Whole antibiotic-killed classic Pseudomonas aeruginosa organisms elicited human lymphocyte [3H]thymidine (TdR) uptake in vitro after 5 days in culture. However, high concentrations of the same preparation did not elicit [3H]TdR incorporation. The investigation of this lymphocyte unresponsiveness revealed that a high dose of P. aeruginosa, when added to lymphocyte cultures together with optimal concentrations of lymphocyte activators (e.g., plant lectins or whole killed Staphylococcus aureus Cowan 1), caused a potent, nonspecifically expressed inhibition of lymphocyte [3H]TdR uptake in response to these mitogens. High doses of P. aeruginosa were not cytotoxic to lymphocytes, and the inhibition caused was reversed when lymphocytes were washed free of bacteria. The inhibition of [3H]TdR uptake by high-dose P. aeruginosa did not require the generation of adherent suppressor cells or prostaglandin-mediated, steroid-sensitive or radiation-sensitive suppressor mechanisms. At optimal lymphocyte stimulatory concentrations of P. aeruginosa, the addition of indomethacin or the depletion of adherent cells caused an increase in lymphocyte [3H]TdR incorporation. This is consistent with an adherent-cell population regulating [3H]TdR uptake in response to P. aeruginosa via a prostaglandin-dependent pathway. This population was not involved in the inhibition of lymphocyte [3H]TdR uptake by high concentrations of P. aeruginosa.

Pseudomonas aeruginosa is an extracellular Many chronic infections are accompanied by in vivo and in vitro impairments of cellular gram-negative organism with immunosuppresimmune function. Absent delayed hypersensitiv- sive capabilities. In vivo, in mice, if adminisity, delayed allograft rejection, and increased tered at 24 h before Listeria sp. infection, killed susceptibility to infection with opportunistic in- P. aeruginosa organisms suppress immunity to tracellular pathogens have been reported in pa- Listeria sp. (21). The infection of mice with P. tients with leprosy (11), tuberculosis (39, 42), aeruginosa also depresses contact sensitivity to fungal infections (3, 9, 20), or parasitic infections oxazolone through the activation of a suppressor (10). Peripheral blood lymphocytes from pa- cell which blocks the afferent arm of this cellular tients with these and other chronic infections are immune response (2, 7). In humans, Pseudomooften unresponsive in in vitro assays of blasto- nas sepsis delays skin homograft rejection (34). genesis, some specifically to antigens from the Cystic fibrosis patients with chronic progressive infecting organism and others to a wider variety pulmonary infections with P. aeruginosa have of activators (3, 4, 12, 19, 20, 23, 33). impaired in vitro lymphocyte proliferation in Several mechanisms of immunosuppression response to Pseudomonas sp. and other grammay be active in chronic infection. Bacteria or negative bacteria (29, 31). These defects in celltheir products may exert direct toxic or inhibi- mediated immunity are apparently acquired as a tory effects upon lymphocytes or monocytes. result of Pseudomonas infection as they imInfecting organisms also may generate suppres- prove with antibiotic therapy (30). sor cells, which in turn inhibit responding or In vitro, normal adult lymphocytes only incorantigen-presenting cells. Monocytic adherent porate [3H]thymidine (TdR) in response to a suppressor cells or T-suppressor cells have been limited concentration range of killed P. aerugindemonstrated in the blood of patients with tuber- osa. Supraoptimal bacterial concentrations do culosis (4, 14), fungal infections (32, 33), and not elicit lymphocyte DNA synthesis (25, 31). This phenomenon of lymphocyte unresponsiveparasitic infections (21, 23). t Present address: Department of Medicine, Mount Sinai ness to high doses of P. aeruginosa may provide an in vitro analog to the impairment of cellular Hospital, New York, NY 10029. 630

VOL . 39, 1983

P. AERUGINOSA-INDUCED IMMUNOSUPPRESSION

immunity in chronic or overwhelming Pseudomonas infections. In this paper, we investigate the specificity and mechanism of unresponsiveness to high-dose P. aeruginosa of normal human lymphocytes. MATERIALS AND METHODS Blood donors. Venous blood (20 to 100 ml) was drawn into heparinized syringes (5 U of heparin per ml of blood) from clinically healthy young adult volunteers, aged 20 to 40. Bacterial and phytomitogen preparations. A clinical

isolate of P. aeruginosa P3 (international serotype 6; supplied by M. J. Thomasson, Case Western Reserve University) was used in all experiments (36). This classic P. aeruginosa strain produces both exotoxin-A and proteases. The immunological reactivity of normal adult peripheral blood lymphocytes for this strain was comparable to that found for several other clinical isolates of P. aeruginosa tested in our laboratory. A Cowan 1 strain of Staphylococcus aureus (derived from American Type Culture Collection strain 12598) was used as a lymphocyte mitogen in these studies. This strain, after antibiotic killing as described below, is a potent mitogen for normal human adult peripheral blood lymphocytes (H. R. Rubin, Ph.D. thesis, Case Western Reserve University, Cleveland, Ohio, 1982). Additional bacterial strains used where indicated were an Escherichia coli strain (ATCC 25922) and Pseudomonas cepacia (strain 715j, a clinical isolate from a cystic fibrosis patient). Bacteria were prepared by a modification of a previously described method (29). Pure bacterial strains were grown under optimal conditions in Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy broth without glucose and killed either by the addition of gentamicin or by incubation in 10%o Formalin at pH 7 overnight (35). After killing by either method, bacteria were washed by repeated centrifugation in sterile isotonic saline and diluted to 10%o (vol/vol) in saline. These stock suspensions were stored at 4°C. On the day of addition to cultures, the supernatant was discarded, the bacterial pellet was suspended in fresh saline to 10%o, and 1, 0.1, 0.01, and 0.001% dilutions were prepared. Antibiotic-killed bacteria were prepared every 2 months from the original strains and tested before use with normal lymphocytes of known reactivity. Formalin-killed bacteria were utilized only where indicated; all other experiments were with antibiotic-killed bacteria. Plant lectins were diluted on the day of use: phytohemagglutinin (PHA; Difco Laboratories, Detroit, Mich.) from a stock solution stored at 4°C to 150, 75, 37, and 19 ,ug/ml, concanavalin A (ConA; Pharmacia Fine Chemicals, Piscataway, N.J.) from a stock solution stored at 4°C to 1,000, 500, 250, and 125 pLg/ml, and pokeweed mitogen (PWM; GIBCO Laboratories, Grand Island, N.Y.) from the undiluted stock solution stored at -20°C to 1:5, 1:10, and 1:20 dilutions. Phytomitogen stock solutions were monitored with lymphocytes of known reactivity and maintained their potency throughout the experimental period. There was a small individual variation in the optimal bacteria or lectin concentrations activating lymphocytes from different individuals. Final concentrations of 0.01% P. aeruginosa, 0.1% S. aureus, 3.75 Fg of

631

15

10

0

2

4

e

a

10

1

2

DAYS IN CULTUW

FIG. 1. Kinetics of lymphocyte proliferation in response to P. aeruginosa: effect of P. aeruginosa concentration. Symbols: 0, 0.1% P. aeruginosa; *, 0.01% P. aeruginosa; A, 0.001% P. aeruginosa. PHA per ml, 25 Fg of ConA per ml, and 1:100 PWM were optimal for most individuals. In all experiments, 0.1% P. aeruginosa was a supraoptimal concentration. Cell preparations. Peripheral blood mononuclear cells containing approximately 85 to 95% lymphocytes were isolated from heparinized peripheral blood by Ficoll-Hypaque centrifugation (37). When indicated, the depletion of adherent cells was performed by plastic adherence (24). Mononuclear cells were suspended at 106 cells per ml in RPMI 1640 medium containing 10 to 20%o heat-inactivated autologous plasma, and 20-ml samples of this suspension were placed in plastic petri dishes 100 mm in diameter. After incubation at 37°C for 1 h, nonadherent cells were decanted. These cells were then incubated on fresh petri dishes for 1 h at 37°C. Nonadherent cells decanted after the second incubation were adherence-depleted cells. The percentage of monocytes in cell preparations was determined by nonspecific esterase staining (41). The percentage of viable cells in these cell preparations was determined by acridine orange-ethidium bromide staining (16). When indicated, mononuclear cells were irradiated in a WCo-irradiator. Mononuclear cells suspended in RPMI 1640 at 5 x 106 cells per ml were placed in sterile plastic test tubes (12 by 75 mm; Falcon Plastics, Oxnard, Calif.), or 10 x 106 to 15 x 106 cells were placed into 50-ml conical-bottomed polystyrene centrifuge tubes (Corning Glass Works, Corning, N.Y.) and irradiated with 0, 500, 1,000, 1,500, or 3,000 rads (as indicated) at 2,000 rads per min. High-dose P. aeruginosa-primed cells and preincubated unstimulated control cells were prepared as follows. Mononuclear cells were suspended at 106/ml as usual (see below). A 0.1- to 0.15-ml amount per tube was placed into 50-ml polystyrene conical-bottomed centrifuge tubes (Corning Glass Works, Corning, N.Y.), and 1 or 0.1% P. aeruginosa was added in the appropriate volume to result in final concentrations of 0.1 or 0.01% P. aeruginosa. This concentration represented an approximate bacteria-to-lymphocyte ratio of 60:1. No or 0.1% P. aeruginosa was added to control tubes. Cells were incubated for 24 to 48 h at 37°C in 5% CO2. They were then washed three times by centrifugation at 800 g for 10 min and suspended as usual for

632

INFECT. IMMUN.

RUBIN ET AL.

TABLE 1. Effect of prior irradiation on lymphocyte [3H]TdR uptake in response to PHA and P. aeruginosa at 5 days of culture Irradi[3H]TdR uptake (cpm) with:

8' 7'

n=10

ation

6I.

2

5-

42 3-

PHAa 0.01% PAb (rads) No additions 0 1,285 ± 1,061C 5,277 ± 1,497c 14,141 ± 1,309 500 159 ± 74 18,436 ± 412 1,527 ± 1,539d 1,000 371 ± 40 7,293 ± 945 -289 ± 25 2,000 374 ± 129 6,029 ± 1,146 -111 ± 50 3,000 65 ± 35 7,229 ± 1,300 7 ± 20 PHA at the optimal concentration for lymphocyte O.5 PC\< \\ [3H]TdR uptake, 3.75 ,ug/ml. b PA, P. aeruginosa. c Results are expressed as the mean the standard deviation of the counts per minute of the [3H]TdR e uptake of triplicate cultures measured on day 5 of culture; net counts per minute are given for cultures .0 1 containing 1 PHA five and hundred P. aeruginosa. Zero versus rads; P < 0.05.

0.05'a

/' I .L

2-

±

1-

I

bkgd. .000 1

P.

.00 1

SBOONC TRATION (%

FIG. 2. Enhancement of responses to optimal and suboptimal but not supraoptimal P. aeruginosa concentrations by adherent-cell depletion. Symbols: 0, [3H]TdR incorporation at 5 days by mononuclear cells; 0, [3H]TdR incorporation at 5 days by plastic-adherent-cell-depleted mononuclear cells.

plating (see below). Cells preincubated with 0.1% P. aeruginosa are referred to as high-dose P. aeruginosaprimed cells (HPC). HPC contained fewer than 3% nonspecific esterase-positive cells. Interleukin-2-enriched media. An interleukin-2-enriched culture medium, prepared as described by Ruscetti and Gallo (26), was kindly provided by Jill Pugh, Immunology Laboratory, Department of Pediatrics, Case Western Reserve University. Interleukin-2 activity was determined by Mary Hilfiker, Immunology Research Laboratory, Cleveland Clinic Foundation, using a cloned mouse cytotoxic T-cell line as described by Gillis et al. (8). A single lot of culture medium with high interleukin-2 activity (423 U/ml) was used in the experiments described in this paper. Assay of lymphocyte DNA synthesis. Lymphocyte culture methods used were similar to those employed in previous studies of lymphocyte responses to whole antibiotic-killed bacteria (25, 28, 29, 31). Lymphocyte microcultures were set up in 96-well flat-bottomed microculture plates (Falcon Plastics), each well containing 10' cells in 0.1 ml of RPMI 1640 supplemented with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (Microbiological Associates, Bethesda, Md.), penicillin, streptomycin, and 20% heat-inactivated autologous plasma (hereafter referred to as supplemented RPMI 1640). Autologous plasma was used to preserve individual variations in responsiveness because extensive previous experiments have shown that using pooled homologous plasma (with or without antibodies to P. aeruginosa) may change individual lymphocyte responses to P. aeruginosa in different directions (29). A 10-p1 volume of the appropriate bacterial dilution or phytomitogen dilution was added to each well. Ten microliters of a 100-, 10-, or 1-

,ug/ml dilution of indomethacin or 10 ,ul of a 10-1, 10-3, 10-5, 10-6, or 10-7 M dilution of hydrocortisone succinate (Solu-Cortef; The Upjohn Co., Kalamazoo, was added at this point when used. Triplicate cultures were set up for every experimental condition. Cultures were incubated at 37°C in 5% CO2 for the indicated number of days (3 days for cultures containing phytomitogens and 5 days for cultures containing bacterial preparations except in kinetics experiments). Five hours before the end of the incubation period, 0.5 p.Ci of [3H]TdR (specific activity, 25 Ci/mmol) was added to each culture in a 10-ptl volume. The cells were harvested with a lymphocyte microharvester (Otto Hiller Co., Madison, Wisc.). The incorporation of [3H]TdR was measured with a Searle Isocap 300 liquid scintillation counter. Statistical methods. The mean of the triplicate cultures was calculated for every condition in each experiment. The net response of an individual to a bacterial preparation, phytomitogen, or combination describes the mean of stimulated cultures minus the mean of unstimulated culture cells from the same individual. Significance was assessed by the two-tailed paired Student t test.

Mich.)

RESULTS

Optimal concentrations of antibiotic-killed P. aeruginosa (0.01 and 0.001%) caused increased peripheral blood lymphocyte [3H]TdR uptake when included in 5-day cultures. In contrast, a high concentration (0.1%) did not induce a proliferative response after 5 days and indeed often significantly inhibited the background proliferative activity of mononuclear cell cultures. Figure 1 illustrates the kinetic curve for lymphocyte [3H]TdR incorporation in response to P. aeruginosa. The decreased proliferation seen at 5 to 6 days in cultures with 0.1% P. aeruginosa was occasionally preceded by a small burst of proliferation at 1 to 4 days, peaking at least 2 days before the proliferation induced by lower, opti-

VOL. 39, 1983

P. AERUGINOSA-INDUCED IMMUNOSUPPRESSION

mal concentrations of P. aeruginosa. High P. aeruginosa concentrations uniformly elicited little proliferation. However, the earlier peak of [3H]TdR uptake was not seen in every kinetic study. To evaluate the possible cytotoxic effect of 0.1% P. aeruginosa, mononuclear cells at 106/ml were incubated for 5 days with 0.1% P. aeruginosa and counted daily to determine recovery and viability. The recovery on days 1 through 5 of cells exposed to 0.1% P. aeruginosa did not differ significantly from the recovery of those exposed to 0.001% P. aeruginosa or of cells incubated without P. aeruginosa. A total of 95% (±5%) of the recovered cells in all samples were viable by acridine orange-ethidium bromide fluorescence. The presence of an early burst of lymphocyte [3H]TdR incorporation in some temporal kinetic studies suggested that high-dose P. aeruginosainduced unresponsiveness is a result of the activation of suppressor cells. To test this hypothesis, several manipulations were performed to selectively affect particular mononuclear cell subsets to determine whether these subsets of cells were necessary for the expression of P. aeruginosa-induced lymphocyte unresponsiveness. Monocytic adherent suppressor cells have been demonstrated to be present in the blood of patients with chronic infections (4, 14, 21, 23, 32, 33, 38) and are necessary for the suppression of lymphocyte mitogenesis by other bacterial products (5, 15). To determine whether monocytic adherent suppressor cells mediate unresponsiveness to P. aeruginosa, the mononuclear cell population was depleted of adherent cells by plastic adherence, resulting in a reduction of nonspecific esterase-positive cells from 15 ± 5% to 4 ± 2%. This depletion significantly increased lymphocyte [3H]TdR uptake in response to optimal and suboptimal concentrations of P. aeruginosa (Fig. 2). However, it had no effect on

[3H]TdR uptake by lymphocytes exposed to high-dose P. aeruginosa. Prostaglandin-mediated indomethacin-sensitive pathways, usually associated with adherent suppressor cells, have been implicated in the suppression of chronic infection and in immunosuppression by bacterial products (5, 15, 21). To determine whether prostaglandin-mediated mechanisms participate in lymphocyte unresponsiveness to P. aeruginosa, 1 jxg of indomethacin per ml was added to lymphocyte cultures. This enhanced responses to P. aeruginosa (P < 0.0025) in the same pattern as monocyte depletion. Again, it enhanced [3H]TdR uptake in response to optimal and suboptimal concentrations of P. aeruginosa but did not affect cultures containing 0.1% P. aeruginosa. The indometh-

18s 16-

+

14-

12a 10 0. 0

X

+.

8 6. 4.

2 I

633

I

[.1 I

I

I

I

r-

m6m.J.

PWM PA PA PA PA bkgd. 1:200 0.01% 0.1% 0.01% 0.1% + PWM PWM 1:200 1:200 FINAL CONCENTRATIONS OF ACTIVATORS FIG. 3. Inhibition of background and PWM-induced lymphocyte proliferation by high- (0.1%) but not optimal- (0.01%) dose P. aeruginosa. Error bars indicate the standard error of the mean.

acin treatment of adherence-depleted mononuclear cells, in two experiments, did not further enhance [3H]TdR uptake in response to optimal P. aeruginosa (data not shown). Steroid-sensitive suppressor mechanisms have been shown in vitro to affect polyclonal antibody responses to PWM (6). In vivo, in humans, steroids depress suppressor T-cell function (27). To see whether steroid-sensitive cells mediate unresponsiveness to high-dose P. aeruginosa, we added hydrocortisone succinate to cultures over a large concentration range. At high doses (over 10-5 M), hydrocortisone depressed lymphocyte [3H]TdR uptake in response to all concentrations of P. aeruginosa. At low doses (10-6 through 10-8 M), hydrocortisone did not affect lymphocyte [3H]TdR uptake in response to P. aeruginosa (data not shown). Radiosensitive T-suppressor cells are induced by bacterial lipopolysaccharide (18). To see whether similar suppressor cells contribute to P.

aeruginosa-induced lymphocyte unresponsiveness, mononuclear cells were irradiated before culture. Prior irradiation with 500 rads or more decreased lymphocyte [3H]TdR uptake in response to all concentrations of P. aeruginosa at 5 days in culture (Table 1). The peak of (3H]TdR uptake in response to PHA was observed at 3 days, with a lower residual response after 5 days in culture. In contrast to the radiosensitivity of the lymphocyte response to P. aeruginosa, the residual responses to PHA at 5 days were enhanced by prior irradiation with 500 and even 3,000 rads.

634

INFECT. IMMUN.

RUBIN ET AL.

The absence of demonstrable suppressor cells mediating high-dose unresponsiveness to P. aeruginosa suggested that high-dose P. aeruginosa-induced unresponsiveness represents the nonspecific inhibition of lymphocyte DNA synthesis rather than the immunologically mediated suppression of the response to P. aeruginosa. To test this possibility, an optimal concentration of one of several other lymphocyte activators (PWM, ConA, PHA, or antibiotic-killed S. aureus Cowan 1) was combined with suboptimal, optimal, and supraoptimal P. aeruginosa concentrations in 3- or 5-day cultures. One-tenth percent P. aeruginosa consistently eliminated 5day lymphocyte [3H]TdR uptake induced by an optimal dose of PWM (Fig. 3) or S. aureus Cowan 1 (Table 2), whereas optimal doses of P. aeruginosa did not. High P. aeruginosa concentrations also greatly decreased lymphocyte [3H]TdR uptake in response to ConA and PHA at 3 days of culture (data not shown). To see whether this inhibitory effect of P. aeruginosa was unique, high doses of other bacterial species were used in parallel experiments. In three experiments, the effect of 0.1% P. aeruginosa on ConA-stimulated mitogen responses at 3 days was compared with the effect of the same concentrations of S. aureus Cowan 1, E. coli, and P. cepacia. Results were as follows (± the standard error of the mean): ConA alone, 82,006 ± 6,936 cpm; ConA and P. aeruginosa, 409 ± 251 cpm; ConA and S. aureus Cowan 1, 60,059 ± 6,379 cpm; ConA and E. coli, 74,292 ± 4,819 cpm; ConA and P. cepacia, 33,032 ± 7,822 cpm. To examine whether this nonspecifically expressed suppression by P. aeruginosa of lymphocyte thymidine uptake was mediated by suppressor cells, the manipulations described above (adherence depletion, irradiation, addition of indomethacin, and addition of hydrocortisone) were repeated to see whether they abrogated the inhibition. Table 3 demonstrates that neither monocyte depletion nor indomethacin affected the inhibition by high-dose P. aeruginosa of lymphocyte [3H]TdR uptake in response to PHA or ConA at 3 days of culture. Hydrocortisone in 10-5 to 10-7 M concentrations did not affect lymphocyte [3H]TdR incorporation in response to PWM in the presence of 0.1% P. aeruginosa. Prior irradiation of mononuclear cells with 500 rads also did not affect the suppression by 0.1% P. aeruginosa of PHA-induced lymphocyte [3H]TdR uptake at 3 days (data not shown). To determine whether the effect of high-dose P. aeruginosa is reversible, mononuclear cells were preincubated with 0.1% P. aeruginosa for 24 to 48 h. Immediately after preparation, these HPC did not incorporate more [3H]TdR than did control cultures kept in the presence of P.

TABLE 2. Inhibition of S. aureus-induced [3H]TdR uptake by high-dose but not optimal-dose P. aeruginosa Addition Uptake SAC1a ........................... 47,464 ± 9,882b SAC1 + 0.01% PAC ................ 55,343 ± 7,172 SAC1 + 0.1% PA ................. 864 + 361d a SAC1, S. aureus Cowan 1 at the concentration eliciting the highest stimulation in the absence of added P. aeruginosa. b Results are expressed as the mean ± the standard error of the mean of the net counts per minute for [3H]TdR uptake of triplicate cultures in five experiments.

c PA, P. aeruginosa. d SAC1 versus SAC1 + 0.1% P. aeruginosa and SAC1 + 0.01% P. aeruginosa versus SAC1 + 0.1% P. aeruginosa; P < 0.0025 in the one-tailed paired Stu-

dent t test.

aeruginosa. After 5 additional days of incubation without the addition of antigen or mitogen, the same HPC, compared with cells preincubated without P. aeruginosa, in five of five experiments spontaneously incorporated a significantly greater amount of [3H]TdR (for HPC, 15,427 ± 7,257 cpm of [3H]TdR incorporated; for preincubated unstimulated cells, 2,680 ± 1,510 cpm of [3H]TdR incorporated). Similarly, when another lymphocyte activator was added to highdose P. aeruginosa-primed cells after the washout of P. aeruginosa, these primed cells always incorporated additional [3H]TdR over the background (Table 4). Thus, the nonspecific inhibition by high-dose P. aeruginosa of lymphocyte [3H]TdR incorporation was reversible and dependent upon the continued presence of P. aeruginosa in the culture. The renewed [3H]TdR uptake by cells preincubated with 0.1% P. aeruginosa after washing and suspension in fresh medium suggested several explanations for the effect of P. aeruginosa on lymphocytes which did not involve the direct inhibition of lymphocyte proliferation. One possibility was the consumption of nutrients in the culture medium by P. aeruginosa. To determine whether nutrients were consumed by killed P. aeruginosa, inhibitory concentrations of formalinized P. aeruginosa were incubated in culture medium alone and in the presence of lymphocytes. After 3 days of incubation, supernatants were removed and used as culture medium for additional 3-day cultures of fresh peripheral blood lymphocytes stimulated with ConA. No significant differences were observed between lymphocyte proliferative responses to ConA in control culture medium and lymphocyte responses in 3-day supernatants from P. aerugino-

VOL. 39, 1983

P. AERUGINOSA-INDUCED IMMUNOSUPPRESSION

635

TABLE 3. Effect of adherent-cell depletion and indomethacin on high-dose P. aeruginosa-induced inhibition of lymphocyte [3H]TdR uptake in response to plant lectins at 3 days of culture

[3H]TdR incorporation (cpm) in: Plant lectin

Ca MNC~

Adherencedepleted cells

MNC + indomethacinb 50,923 + 11,210 332 ± 398f

29,578 ± 10,979 58,482 + 10,531d ConAc 557 ± 263 ConA + PA' 436 ± 24 76,939 ± 13,627 99,516 ± 23,021 PHA 108,805 ± 17,041 559 ± 334 791 ± 377f PHA + PA 1,316 ± 717f a MNC, Mononuclear cells. b Indomethacin at the final concentration in a culture of 1 Fg/ml. ConA and PHA at the optimal stimulatory concentration in these experiments, 50 and 5 Rg/ml, respectively. d Results are expressed as the mean ± the standard deviation for two experiments of net counts per minute of lymphocyte [3H]TdR incorporation on day 3 of culture, assayed in triplicate cultures in each experiment for each

condition. ' P. aeruginosa at 0.1% vol/vol concentration, Formalin-killed. f ConA versus ConA + P. aeruginosa and PHA versus PHA + P. aeruginosa; P < 0.05 in the one-tailed paired student t test.

sa with or without lymphocytes (data not

shown). A second explanation for the inhibition of mitogenic responses by P. aeruginosa was the consumption of interleukin-2 by a rapidly dividing cell population early in the culture period. Insufficient interleukin-2 might then remain to support later mitogenic responses. In one set of experiments, peripheral blood mononuclear cells were suspended directly in interleukin-2enriched culture media and then exposed to ConA and supraoptimal concentrations of P. aeruginosa. In other experiments, cells suspended in fresh media were exposed to ConA and P. aeruginosa, and interleukin-2-enriched medium was added to a 40% concentration at 0 and 24 h. In all experiments, the inhibition of mitogenic responses by P. aeruginosa was not renewed by interleukin-2 (data not shown). Yet another explanation for the observed effect of a supraoptimal dose of P. aeruginosa might be the inhibition of [3H]TdR incorporation, without the inhibition of lymphocyte proliferation. To determine whether P. aeruginosa interferes with [3H]TdR incorporation, peripheral blood cell cultures were stimulated with ConA and incubated for 3 days. Supraoptimal concentrations of P. aeruginosa were added after 3 days, 1 h before [3H]TdR was added to the cultures. In three such experiments, ConAstimulated cells with P. aeruginosa added at 3 days incorporated 77,071 ± 9,973 cpm; with P. aeruginosa added at 0 h, they incorporated 931 ± 406 cpm; cells stimulated with ConA in the absence of P. aeruginosa incorporated 105,598 ± 14,115 cpm. DISCUSSION P. aeruginosa has been shown to inhibit immune response via several mechanisms. B sup-

pressor lymphocytes have been described to mediate the suppression by P. aeruginosa infection of the afferent arm of contact sensitivity to oxazolone in mice (2, 7). Killed P. aeruginosa have also been demonstrated to inhibit cellular immunity to Listeria sp. and delayed hypersensitivity to sheep erythrocytes via a splenic, adherent, macrophage-like cell (22). In contrast, our results demonstrated a nonspecific, potent inhibition of lymphocyte DNA synthesis by high-dose P. aeruginosa. This inhibition did not involve cytotoxicity and, indeed, was reversible after 48 h of exposure to high-dose P. aeruginosa by the removal of bacteria. Adherent monocytic, indomethacin-sensitive, steroid-sensitive, and radiosensitive cells apparently did not mediate this effect. The reversibility of this inhibition and the lack of evidence for suppressor cell participation in it suggest a direct inhibition by high-dose killed P. aeruginosa of [3H]TdR uptake by responding lymphocytes. Because the removal of P. aeruginosa resulted in spontaneous [3H]TdR uptake without the addition of another activator, the block of DNA synthesis by high-dose P. aeruginosa occurred at a point in the activation process which followed the initiation by P. aeruginosa of an irreversible activating signal. The block probably preceded a step in the activation process responsible for further amplification and recruitment (e.g., secretion of interleukin-2), because after removal, [3H]TdR incorporation was first very low, but by 5 days later was quite significant. This apparently direct inhibition of lymphocyte [3H]TdR uptake or DNA synthesis resembles inhibition by high-dose ConA (1, 17) in its reversibility and in its apparent independence from suppressor cell activity. Other microbial products have been noted to inhibit lymphocytes directly. Hatching fluid from Schistosoma man-

636

INFECT. IMMUN.

RUBIN ET AL.

TABLE 4. Effect of preincubation with high-dose P. aeruginosa on lymphocyte [3H]TdR uptake in response to ConA and S. aureus Cowan 1 [3H]TdR uptake (cpm)

MitogenaMNbHC HpCb MNCb + S. aureus

27,328 ± 13,000c

23,472 ± 1,881

Cowan 1 + ConA a Results aureus

22,938 ± 6,187 27,092 ± 12,313 are reported for the concentration of S. Cowan 1 on ConA eliciting the optimal re-

in each individual. The response to S. aureus measured at 5 days in culture, and the response to ConA was measured at 3 days. b MNC, Mononuclear cells preincubated in flasks for 48 h and washed; HPC, mononuclear cells preincubated for 48 h with 0.1% P. aeruginosa and washed. the standard c Results are expressed as the mean error of the mean for three experiments of net counts per minute of lymphocyte [3H]TdR incorporation (cpm in stimulated cultures minus cpm in unstimulated cultures). sponse

was

±

soni eggs exerts a direct dose-dependent inhibitory effect upon lymphocyte blastogenesis without cytotoxicity and apparently without suppressor cell activity (40). Direct lymphocyte inhibition by microbial products thus may occur in the presence of high local antigen concentrations and may be a factor which enables an organism to cause chronic infection. All P. aeruginosa strains tested in our laboratory, including strains selected for very low exotoxin-A or very low protease production, are potent lymphocyte inhibitors (R. U. Sorensen and J. D. Klinger, unpublished data). Although the stimulatory activity of many other bacterial preparations and activators often diminishes at high concentrations, quantitative comparisons with other bacteria, including P. cepacia, E. coli, and S. aureus, indicate that only P. aeruginosa is markedly inhibitory for lymphocyte proliferation. P. aeruginosa exotoxin-A decreases lymphocyte thymidine uptake, but in parallel with decreased protein synthesis and with cell death (13). Therefore, it is unlikely to be involved in the inhibition reported here, which was not associated with cytotoxicity. The component of P. aeruginosa responsible for the remarkable suppressive activity which we observed is now under investigation, and studies to characterize it are being reported separately. Our data also demonstrated the presence in normal mononuclear cell populations of adherent, presumably monocytic, cells which regulated [H]TdR uptake in response to optimal and suboptimal P. aeruginosa concentrations. Indomethacin also enhanced responses to optimal and suboptimal P. aeruginosa concentrations, demonstrating that a prostaglandin-mediated

pathway also may regulate human lymphocyte [3H]TdR uptake in response to P. aeruginosa. The similarity of the pattern of enhancement by these two manipulations, as well as preliminary experiments demonstrating that prior adherence depletion abrogates the effect of indomethacin, suggests that these two manipulations affect the same prostaglandin-secreting adherent suppressor cell population. Similar cells mediate suppression by other bacterial products (5, 15), and are present in the blood of patients with chronic infections (21, 23, 32, 33). An increase in these cells may be another mechanism of unresponsiveness to P. aeruginosa in patients chronically infected with this organism. P. aeruginosa is the usual pathogen in chronic progressive lung infections of patients with cystic fibrosis. Those patients who are most severely infected and who eventually die of their infections are also those with low [3H]TdR incorporation in response to P. aeruginosa and other gram-negative bacteria (29-31). This impairment of cellular immunity, detected in the peripheral blood of patients with cystic fibrosis, probably represents an immunoregulatory disturbance secondary to chronic infection. It differs from the high-dose inhibition described in this paper in that cystic fibrosis peripheral blood lymphocytes have normal proliferative responses to mitogens, even in the presence of cystic fibrosis plasma. The nonspecific inhibition caused by high doses of P. aeruginosa may paralyze cell-mediated immune reactions in the infected lung environment. Such a possibility is supported by the concomitance of severe, progressive P. aeruginosa bronchitis and very low susceptibility to systemic infection in patients with cystic fibrosis. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (HL-24244 and A1-14862) and a grant from the Cystic Fibrosis Foundation. Haya Rubin, an M.D./Ph.D. student at Case Western Reserve University, was supported by the National Institutes of Health Medical Scientist Training Program. LITERATURE CITED 1. Andersson, J., 0. Sjoberg, and G. Moller. 1972. Reversibility of high dose unresponsiveness to concanavalin A in thymus lymphocytes. Immunology 23:637-646. 2. Colizzi, V., C. Garzelli, M. Campa, and G. Falcone. 1978. Depression of contact sensitivity by enhancement of suppressor cell activity in Pseudomonas aeruginosa-injected mice. Infect. Immun. 21:354-359. 3. Cox, R. A., and J. R. Vivas. 1977. Spectrum of in vivo and in vitro cell-mediated immune responses in coccidioidomycosis. Cell. Immunol. 31:130-141. 4. Ellner, J. J. 1978. Suppressor adherent cells in human tuberculosis. J. Immunol. 121:2573-2579. 5. Ellner, J. J., and P. J. Spagnuolo. 1979. Suppression of antigen and mitogen induced human T lymphocyte DNA synthesis by bacterial lipopolysaccharide: mediation by

VOL. 39, 1983

P. AERUGINOSA-INDUCED IMMUNOSUPPRESSION

monocyte activation and production of prostaglandins. J. Immunol. 123:2689-2695. 6. Fauci, A. S., K. R. Pratt, and G. Whalen. 1977. Activation of human B lymphocytes. IV. Regulatory effects of corticosteroids on the triggering signal in the plaque-forming cell response of human peripheral blood B lymphocytes to polyclonal activation. J. Immunol. 119:598-603. 7. Garzelli, C., V. Colizzi, M. Campa, L. Bozzi, and G. Falcone. 1979. Depression of contact sensitivity by Pseudomonas aeruginosa-induced suppressor cells which affect the induction phase of immune response. Infect. Immun. 26:4-11. 8. Gillis, S., M. M. Ferm, W. Ou, and K. A. Smith. 1978. Tcell growth factor: parameters of production and quantitative microassay for activity. J. Immunol. 120:2027-2032. 9. Graybill, J. R., and R. H. Alford. 1974. Cell-mediated immunity in cryptococcosis. Cell. Immunol. 14:12-21. 10. Greenwood, B. M., H. C. Whittle, and D. H. Molyneux. 1973. Immunosuppression in Gambian trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 67:846-850. 11. Han, S. H., R. S. Weiser, and S. T. Kau. 1971. Prolonged survival of skin allografts in leprosy patients. Int. J. Lepr. 39:1-6. 12. Han, S. H., R. S. Weiser, and J. J. Tseng. 1971. Lymphotoxin production by lymphocytes from leprosy patients. Int. J. Lepr. 39:719-725. 13. Iglewski, B. H., J. Sadoff, M. J. Bjorn, and E. S. Maxwell. 1978. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc. Natl. Acad. Sci. U.S.A. 75:3211-3215. 14. Katz, P., R. A. Goldstein, and A. S. Fauci. 1979. Immunoregulation in infection caused by Mycobacterium tuberculosis: the presence of suppressor monocytes and the alteraton of subpopulations of T lymphocytes. J. Infect. Dis. 140:12-21. 15. Kleinhenz, M. E., J. J. Ellner, P. J. Spagnuolo, and T. M. Daniel. 1981. Suppression of lymphocyte responses by tuberculous plasma and mycobacterial arabinogalactan. Monocyte dependence and indomethacin reversibility. J. Clin. Invest. 68:153-162. 16. Lee, S. K., J. Singh, and R. B. Taylor. 1975. Subclasses of T cells with different sensitivities to cytotoxic antibody in the presence of anesthetics. Eur. J. Immunol. 5:259-262. 17. McClain, D. A., and G. M. Edehnan. 1976. Analysis of the stimulation-inhibition paradox exhibited by lymphocytes exposed to concanavalin A. J. Exp. Med. 144:14941508. 18. Miller, R. A., S. Gartner, and H. S. Kaplan. 1980. The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum. Cell. Immunol. 55:210-218. 19. Musher, D. M., R. F. Schell, and J. M. Knox. 1974. In vitro lymphocyte response to Treponema refringens in human syphilis. Infect. Immun. 9:654-657. 20. Opelz, G., and M. I. Scheer. 1975. Cutaneous sensitivity and in vitro responsiveness of lymphocytes in patients with disseminated coccidioidomycosis. J. Infect. Dis. 132:250-255. 21. Peterson, E. A., F. A. Neva, C. N. Oster, and H. B. Diaz. 1982. Specific inhibition of lymphocyte-proliferation responses by adherent suppressor cells in diffuse cutaneous leishmaniasis. N. Engl. J. Med. 306:387-392. 22. Petit, J. D., G. Richard, B. Albert, and G. L. Daguet. 1982. Depression by Pseudomonas aeruginosa of two Tcell-mediated responses, anti-Listeria immunity and delayed type hypersensitivity to sheep erythrocytes. Infect. Immun. 35:900-908. 23. Plessens, W. F., S. Ratiwayanto, S. Tuti, J. H. Palmieri, P. W. Piessens, I. Koiman, and D. T. Dennis. 1980. Antigen-specific suppressor cells and suppressor factors in

637

human filariasis with Brugia malayi. N. Engl. J. Med. 302:833-837. 24. Rosenberg, S. A., and P. E. Lipsky. 1979. Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells. J. Immunol. 122:926-931. 25. Rubin, H. R., R. U. Sorensen, and S. H. Pohnar. 1981. Lymphocyte responses of human neonates to bacterial antigens. Cell. Immunol. 57:307-315. 26. Ruscetti, F. W., and R. C. Gallo. 1981. Human T-lymphocyte growth factor: regulation of growth and function of Tlymphocytes. Blood 57:379-393. 27. Saxon, A., R. H. Stevens, S. J. Ramer, P. J. Clements, and D. T. Yu. 1978. Glucocorticoids administered in vivo inhibit human suppressor T lymphocyte function and diminish B lymphocyte responsiveness in in vitro immunoglobulin synthesis. J. Clin. Invest. 61:922-930. 28. Sorensen, R. U., P. A. Chase, R. C. Stern, and S. H. Polmar. 1981. Influence of cystic fibrosis plasma on lymphocyte responses to Pseudomonas aeruginosa in vitro. Pediatr. Res. 15:14-18. 29. Sorensen, R. U., R. C. Stern, P. Chase, and S. H. Polmar. 1979. Defective cellular immunity to gram-negative bacteria in cystic fibrosis patients. Infect. Immun. 23:398-402. 30. Sorensen, R. U., R. C. Stern, P. A. Chase, and S. H. Polnar. 1981. Changes in lymphocyte reactivity to Pseudomonas aeruginosa in hospitalized patients with cystic fibrosis. Am. Rev. Respir. Dis. 123:37-41. 31. Sorensen, R. U., R. C. Stern, and S. H. Polmar. 1977. Cellular immunity to bacteria: impairment of in vitro lymphocyte responses to Pseudomonas aeruginosa in cystic fibrosis patients. Infect. Immun. 18:735-740. 32. Stobo, J. D. 1977. Immunosuppression in man: suppression by macrophages can be mediated by interactions with regulatory T cells. J. Immunol. 119:918-924. 33. Stobo, J. D., S. Paul, R. E. Van Scoy, and P. E. Hermans. 1976. Suppressor thymus-derived lymphocytes in fungal infection. J. Clin. Invest. 57:319-328. 34. Stone, H. H., K. S. Given, and J. D. Martin. 1967. Delayed rejection of skin homografts in pseudomonas sepsis. Surg. Gynecol. Obstet. 124:1067-1670. 35. Teodorescu, M., E. P. Mayer, and S. Dray. 1977. Identification of five human lymphocyte subpopulations by their differential binding of various strains of bacteria. Cell. Immunol. 29:353-362. 36. Thomassen, M. J., C. A. Demko, R. E. Wood, B. Tandler, D. G. Dearborn, B. Boxerbaum, and P. J. Kuchenbrod. 1980. Ultrastructure and function of alveolar macrophages from cystic fibrosis patients. Pediatr. Res. 14:715721. 37. Thorsby, E., and A. Bratalle. 1970. Histocompatibility

testing. Munksgaard, Copenhagen.

38. Todd, C. W., R. W. Goodgame, and D. G. Colley. 1979. Immune responses during human Schistosomiasis mansoni. V. Suppression of schistosome antigen-specific lymphocyte blastogenesis by adherent/phagocytic cells. J. Immunol. 122:1440-1446. 39. Waxman, J., and M. Lockshin. 1973. In vitro and in vivo cellular immunity in anergic miliary tuberculosis. Am. Rev. Respir. Dis. 107:661-664. 40. Wright, E. P., C. D. Guthrie, D. Salim, T. J. 0. Hilditch, and P. K. Das. 1982. Inhibition of lymphocyte activation by hatching fluid from Schistosoma namsoni eggs. Infect. Immun. 36:419-422. 41. Yam, L. T., C. Y. Li, and W. H. Crosby. 1971. Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. SS:283-290. 42. Zeitz, S. J., J. H. Ostrow, and P. P. van Ardsel, Jr. 1974. Humoral and cellular immunity in the anergic tuberculosis patient. J. Allergy Clin. Immunol. 53:20-26.