Vol. 58, No. 12

INFECTION AND IMMUNITY, Dec. 1990, p. 3856-3862

0019-9567/90/123856-07$02.00/0 Copyright © 1990, American Society for Microbiology

Immune Suppression Induced by Actinobacillus actinomycetemcomitans: Effects on Immunoglobulin Production by Human B Cells BRUCE J. SHENKER,* LAURA A. VITALE, AND DEBORAH A. WELHAM Department of Pathology, University of Pennsylvania School of Dental Medicine, 4010 Locust Street, Philadelphia, Pennsylvania 19104-6002 Received 27 December 1989/Accepted 3 September 1990

AcfinobaciUus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.

philic bacterium, is a suspected etiologic agent in juvenile periodontitis. Additionally, this organism has been identified in blood samples from patients with bacterial endocarditis, meningitis, and abscesses of the jaw and face (4, 11, 25, 30, 46). We have shown that extracts prepared from several different A. actinomycetemcomitans strains contain a heatlabile immunosuppressive factor (ISF) that is capable of inhibiting DNA, RNA, and protein synthesis in lymphocytes activated by both mitogens and antigens (38). These inhibitory effects were not associated with altered cell viability. In this study we have extended our earlier observations and demonstrate that A. actinomycetemcomitans ISF is also capable of inhibiting immunoglobulin production by human B cells.

The virulence of microbes may sometimes be the consequence of their ability to resist, escape, or subvert host defense mechanisms. The ability of microorganisms to evade or suppress the immune response of the host not only affects the course of initial infection by facilitating spread, multiplication, and persistence, but may also lead to enhanced susceptibility to infection by secondary pathogens (reviewed in references 31 and 44). Such modulation of the immune response may be a critical event in the outcome of numerous infections, including measles (1), rubella (22), influenza (34), leprosy (14), candidiasis (19, 28), leishmaniasis (27), trypanosomiasis (2, 26), cryptococcosis (7), tuberculosis (24), and syphilis (41), among others. Perhaps the most prominent example of this relationship between host and pathogen is acquired immune deficiency syndrome, in which the etiologic agent, human immunodeficiency virus, infects and destroys a subpopulation of T lymphocytes. The nature of and the contribution of the immune system to the pathogenesis of periodontal disease are poorly understood. However, recent studies (reviewed in reference 32) suggest that the development and/or expression of these disorders may be associated with immunologic dysfunction. Although the basis for this dysfunction is unclear, we have previously demonstrated that several suspected periodontal pathogens are capable of inhibiting various aspects of lymphoid function (35, 36, 38, 39). One such organism, Actinobacillus actinomycetemcomitans, a gram-negative, capno*

MATERIALS AND METHODS Cell preparation. Human peripheral blood lymphocytes (HPBL) were prepared as described previously (36). Briefly, HPBL were isolated from 100 to 200 ml of heparinized venous blood from healthy donors. The blood was first centrifuged at 300 x g for 15 min at 5°C, the plasma was removed, and the cells were brought back to the original volume with Hanks balanced salt solution (HBSS). HPBL were then isolated by buoyant density centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) by the method of Boyum (3). The HPBL were washed twice with Hanks balanced salt solution (Ca2+ and Mg2+ free) containing 0.5% bovine serum albumin and diluted to 10 x 106 to 20 x 106 viable cells per ml. Viable-cell counts were performed by assessing trypan blue dye exclusion.

Corresponding author. 3856

VOL. 58, 1990

IMMUNE SUPPRESSION BY A. ACTINOMYCETEMCOMITANS

Purified populations of lymphocytes and monocytes were obtained by counterflow centrifugal elutriation as described by Wahl et al. (43) and modified in our laboratory (36).

Briefly, mononuclear cells (lymphocytes and monocytes) obtained as described above; 2 x 108 to 4 x 108 cells placed in 20 ml of HBSS (Ca2' and Mg2+ free) containing 0.5% bovine serum albumin and pumped into a Beckman elutriator rotor (Beckman JE-6 rotor equipped with a standard chamber; Beckman Instruments, Fullerton, Calif.) at a flow rate of 7 ml/min with a rotor speed of 1,960 rpm. This initial flow rate allows stratification of the mononuclear cells in the horizontal chamber according to size and density, while any contaminating platelets are eluted. Cells were then eluted by sequential increases in the flow rate; one fraction of 150 to 200 ml was eluted at each flow rate. Lymphocytes were eluted at 7 to 12 ml/min, while monocytes were eluted at 14 to 16 ml/min. All fractions were counted and monitored for their size profile on a model ZBI Coulter counter connected to an IBM-PC with appropriate hardware and software (Personal Computer Analyzer; Nucleus Inc., Oak Ridge, Tenn.) to allow size distribution analysis. Cell purity was assessed by (i) morphologic appearance, (ii) nonspecific esterase stain, and (iii) immunofluorescence. The lymphocyte preparations were routinely >98% pure and the monocytes were 85 to 95% pure by these were were

criteria. Purified populations of B cells and T cells were obtained by E-rosette formation as described previously (13). Briefly, sheep erythrocytes were washed and treated with 0.14 M 2-aminoethylisothiouronium bromide at pH 9 for 15 min. After four washes, sheep erythrocytes were incubated with 107 lymphocytes (obtained as described above by counterflow centrifugal elutriation) for 60 min as a cell pellet. The cells were then gently resuspended; nonrosetted cells were separated from rosetted cells on Ficoll-Hypaque as described above. Nonrosetted cells found at the interface were >98% free of T cells, and the rosetted cells found in the pellet were lysed to remove sheep erythrocytes and found to contain >98% T cells when stained with anti-CD3 monoclonal antibody (Ortho Pharmaceutical, Raritan, N.J.) and analyzed by flow cytometry. In selected experiments, further separation of B cells from natural killer (NK) cells was achieved by panning (45). The rosette-negative cells were incubated with either anti-CD19 (anti-B cell; Becton Dickinson, Mountain View, Calif.) or anti-CD16 (anti-NK cell) monoclonal antibody and washed, and the appropriate cell population was obtained by removal of the antibody-coated cells on plastic dishes coated with goat anti-mouse immunoglobulin antibody. The resulting nonadherent populations of B cells and NK cells were found to be >95% pure when assessed by flow cytometry. Cell culture. Routine screening of column fractions for ISF activity was performed as described previously (38). Briefly, 0.1 ml of HPBL suspension containing 2 x 105 cells in RPMI 1640 medium (GIBCO, Grand Island, N.Y.) containing 2% human blood type AB serum was placed into each well of flat-bottomed microculture plates (Becton Dickinson). Each culture received 0.1 ml of medium or various concentrations of ISF diluted in medium and sterilized through a filter (0.22-pum pore size; Schleicher & Schuell, Keene, N.H.). The cells were then incubated for 60 min at 37°C, at which time the cultures received an optimal mitogenic dose of concanavalin A (1 gig per culture; Calbiochem, La Jolla, Calif.). The cells were incubated for 96 h, and proliferation (DNA synthesis) was assessed by the incorporation of

[3H]thymidine ([3H]TdR).

3857

Cultures for the measurement of immunoglobulin production were made in 0.2 ml of RPMI 1640 containing 5% fetal bovine serum and antibiotics as described previously (37) in 96-well round-bottomed tissue culture plates (Corning). Each culture contained 1 x 105 T cells, 0.5 x 105 B cells, and 2.5 x 104 monocytes; these conditions were found to be optimal in preliminary experiments. As indicated, cells were exposed to either purified ISF (see below) or medium (control) and, in some experiments, were washed with HBSS prior to incubation. Additionally, all cultures received 25 ,ul of either medium (control) or pokeweed mitogen (PWM; final concentration, 0.2 ,ug/ml; Calbiochem). Cell cultures were incubated for 8 days at 37°C in humidified air containing 5% C02, at which time culture supernatants were collected and pooled from replicate cultures for assay of immunoglobulin content. Assay for immunoglobulin. Culture supernatants were assayed for total secreted immunoglobulin M (IgM) and IgG by an enzyme-linked immunosorbent assay (ELISA) (37). Briefly, flat-bottomed (Falcon 3912; Becton Dickinson) microculture plates were coated with 200 RI of a solution containing either affinity-purified goat anti-human IgG [F(ab')2 fragment-specific; Boehringer Mannheim, Indianapolis, Ind.] or goat anti-human IgM (mu-chain specific; Cooper Biomedical) at 5 jxg of protein per ml in carbonate buffer (pH 9.6, room temperature, 4 h). The plates were washed with phosphate-buffered saline (PBS), and unbound sites were blocked by incubating the wells with 200 pAl of BLOTTO (room temperature, 60 min) and stored at -20°C until further use. For assay, the plates were thawed and washed with PBS, and 100 ,ul of an appropriate dilution of culture supernatant (diluted in 1 M diethanolamine buffer containing 0.5% bovine serum albumin) was added to triplicate wells. Following a 4-h incubation, the wells were washed and the amount of IgG or IgM bound to the wells was determined by the addition of 200 RI of goat anti-human immunoglobulin (IgG, IgM, and IgA; Boehringer Mannheim) conjugated to alkaline phosphatase. The plates were incubated overnight at 5°C and washed, and then color was developed by the addition of 200 ,ul of a solution containing p-nitrophenylphosphate (1 mg/ml; Sigma Chemical Co., St. Louis, Mo.) in 1 M diethanolamine buffer (pH 9.8). After 30 min, 50 ,ul of 2 M NaOH was added to each well to stop the enzymatic reaction. Supernatant (150 ,ul) from each well was transferred to a clean polystyrene microculture plate (Nunc, Roskilde, Denmark), and color development was detected on a Dynatech (Alexandria, Va.) ELISA plate reader. The optical density values were related to immunoglobulin concentration on a standard curve (generated by linear regression analysis) simultaneously prepared with pure IgG or IgM (Cooper Biomedical and Calbiochem, respectively). Culture supernatants were diluted so that their immunoglobulin levels fell within the range of the standard curve (5 to 125 ng/ml). IL-2 production. To assess interleukin-2 (IL-2) production, supernatants were generated in 1-ml cultures containing 2 x 106 pure T cells and 1 x 105 monocytes. Cells were exposed to either medium (control) or various concentrations of ISF and phytohemagglutinin (PHA; 0.8 ,ug/ml; Wellcome Diagnostics, Dartford, England). The supernatants were collected after 24 h of incubation, and cells were removed by centrifugation. IL-2 activity was determined by assessing the ability of the culture supernatants to support the growth of an IL-2-dependent cell line. CTLL-20 cells (1 x 104 cells per well; kindly provided by P. L. Simon, Smith-Kline and French Labs) suspended in RPMI 1640 were incubated for 18

3858

SHENKER ET AL.

h with 100 ,ul of appropriately diluted culture supernatant or various concentrations of IL-2 standard (80 U/ml) diluted in culture medium. [3H]TdR incorporation was assessed as described above. IL-2 activity was determined by comparing [3H]TdR incorporation supported by culture supernatants with that supported by known concentrations of an IL-2 standard. Assessment of IL-2R expression and release of soluble IL-2R. Purified T cells (5 x 106/ml) and monocytes (1 x 105/ml) were incubated for 24 h with PHA (1.0 ,ug/ml) in the presence or absence of ISF. The cells were washed three times with PBS containing 2% fetal bovine serum and stained with anti-human IL-2 receptor (IL-2R) monoclonal antibody (anti-CD25; Becton Dickinson), followed (after washing) by fluorescein-conjugated goat anti-mouse immunoglobulin (Becton Dickinson). The cells were fixed in 1% paraformaldehyde and analyzed on a modified FACS IV flow cytometer equipped with logarithmic amplifiers for fluorescence measurements (8). Cells which gave fluorescence signals brighter than that observed for 95 to 99% of appropriate control cells were considered positive. The mean channel number of the logarithm of the fluorescence intensities was the parameter used to compare the relative density of IL-2R on the various positive populations. In order to measure the release of soluble IL-2R by activated cells, purified T cells were incubated as described above for surface expression of IL-2R. The cultures were harvested at various times, and the replicates were pooled and centrifuged. The proteinase inhibitor phenylmethylsulfonyl fluoride (2 mM) was added to the supernatants, which were either assayed immediately or frozen for future assay. Assay of soluble IL-2R was achieved by ELISA with a commercial immunoassay kit (T Cell Sciences Inc., Cambridge, Mass.). Purification of ISF. A. actinomycetemcomitans 652 was cultured as previously described (38). Briefly, the bacteria were grown for 48 h at 37°C in fluid thioglycolate medium containing 0.4% sodium bicarbonate. Harvested organisms were washed with PBS and extracted in 50 mM Tris buffer, pH 8.0, containing 10 mM NaCl, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 ,ug of lysozyme per ml, and 0.5 pLg of DNase per ml. Following one cycle of freeze-thawing, debris and remaining bacterial cells were removed by centrifugation at 10,000 x g, and the supernatant was ultracentrifuged for 60 min at 100,000 x g. Bacterial extracts were first fractionated by ammonium sulfate precipitation; all activity precipitated between 30 and 55%. Following dialysis against 10 mM Tris buffer, pH 7.0, containing 10 mM NaCl and 1 mM EDTA, the sample was applied to a DEAE-Sephacel column (1.5 by 15 cm; Pharmacia, Uppsala, Sweden) preequilibrated in this buffer. The column was then extensively washed and eluted with a linear NaCl gradient (10 to 400 mM) at a flow rate of 20 ml/h. Four-milliliter fractions were collected and monitored for both A280 and ISF activity (reduction of mitogen-induced [3H]TdR incorporation). Appropriate fractions were pooled and concentrated by membrane ultrafiltration (YM-10 and Centricon 10; Amicon, Lexington, Mass.). Active fractions from DEAE chromatography were pooled and concentrated for further fractionation by gel filtration chromatography on two Superose 12 columns connected in series (Pharmacia). The columns were equilibrated with PBS; 200-,u samples were applied, and 0.5-ml fractions were collected. All fractions were monitored for A280 and for ISF activity. Active fractions were pooled, concentrated by ultrafiltration, and assessed for purity by sodium

INFECT. IMMUN.

-1

2

AB- .. I.

FIG. 1. SDS-PAGE of ISF isolated from A. actinomycetemcomitans 652. Cells were treated with extraction buffer and the ISF was purified as described in Materials and Methods. Lane 1, Crude extract stained with silver; lane 2, purified ISF. Molecular weight standards: A, bovine serum albumin (66,000); B, ovalbumin (45,000); C, carbonic anhydrase (31,000).

dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (16). RESULTS We reported previously that crude sonic extracts of A. actinomycetemcomitans contained a heat-labile, nondialyzable protein capable of inhibiting human T-lymphocyte activation by both mitogens and antigens. Inhibition was reflected in altered DNA, RNA, and protein synthesis (17). We have now purified the ISF to apparent homogeneity (see Materials and Methods for details). As shown in Fig. 1, SDS-PAGE analysis of the purified ISF revealed a single band corresponding to approximately 60 kDa. The purified ISF caused a dose-dependent inhibition of lymphocyte proliferation, as demonstrated in Fig. 2; this represents a >2,000-fold increase in specific activity, with the crude material exhibiting an ID50 (dose required to cause 50% inhibition of [3H]TdR incorporation) of 56.2 ng/ml and the pure ISF preparation exhibiting an ID50 of 0.026 ng/ml. In this study we wanted to determine the ability of ISF to alter immunoglobulin production by human B cells in response to PWM. As shown in Fig. 3, purified ISF caused a dose-dependent inhibition of immunoglobulin production. Production of both IgG and IgM was inhibited with similar 120

-

0 e c

0

100-

*0

0

60z I a:

0

0

40 20--

0 ..

0

.0001

.001

w-_*

.01 .1 1.0 10 CONCENTRATION ISF (ng protein/ml)

.....

100

FIG. 2. Comparison of effects of crude and pure ISF preparations on HPBL proliferation. HPBL were incubated with various amounts of either crude (0) or purified (0) ISF for 60 min, followed by the addition of an optimal mitogenic dose of concanavalin A. [3H]TdR incorporation was measured after incubation for 4 days. The results are plotted as the percentage of [3H]TdR incorporation in control cultures receiving mitogen alone. Each point represents the mean value of quadruplicate cultures in a representative experiment; standard errors (SEs) were within 5% of the mean.

2u0u

3859

IMMUNE SUPPRESSION BY A. ACTINOMYCETEMCOMITANS

VOL. 58, 1990 -

L.

E

0

A

80

+

60

+

1500-

A

c

cJ

z

0E

1000-. A

0 z

0

500-

z

40 +

-J

0

0

0.1 1.0 10.0 100.0 ISF CONCENTRATION (pg protein/ml)

0

0

FIG. 3. Effects of A. actinomycetemcomitans ISF on IgG and IgM synthesis. Cell cultures of purified T cells, B cells, and monocytes were established as described in Materials and Methods. Cells were incubated with medium or various amounts of purified ISF for 60 min, followed by the addition of PWM. After 8 days, culture supernatants were harvested, and the amounts of secreted IgG (A) and IgM (A) were measured by ELISA. Results are plotted as the mean amount produced in triplicate cultures of a representative experiment; standard deviations were within 10%o of the mean.

ID50s of 3 and 4 pg/ml, respectively. It is interesting that immunoglobulin production was found to be significantly more sensitive to the inhibitory effects of ISF than were the proliferative responses of T cells. We reported previously that the ISF was heat labile in its ability to suppress cell proliferation. Similarly, heat treatment (56°C, 30 min) of the ISF also destroyed its ability to suppress immunoglobulin production (data not shown). We next wanted to monitor the kinetics of this inhibition and to determine the conditions that result in maximal suppression. Previous studies indicated that to achieve maximal inhibition of T-cell proliferation, the ISF required a 90-min preincubation period. Figure 4 demonstrates the results of experiments in which ISF was added to cell cultures at various times before and after the addition of mitogen (PWM). Maximal suppression of immunoglobulin production (greater than 85% inhibition) was observed when the cells were exposed to the ISF at any time during the first 3 h of incubation. In contrast to its effect on T cells, no preincubation was necessary for suppression of immunoglobulin production. Addition of ISF at 18 to 24 h or later resulted in reduced suppression; no assessments were made between 3 and 18 h. Preliminary experiments indicated that no effect was observed when ISF was added after 48 h (data not shown). It was also evident that inhibition was not the result of altered cell viability; cells exposed for 8 days in the presence of ISF exhibited 98% of the viability that was observed in control cultures exposed only to PWM. In separate experiments, it was determined that ISF did not have to be present for the entire incubation period. Cells exposed to ISF for 30 min and then extensively washed were inhibited in their ability to produce immunoglobulin (data not shown). These experiments suggest that the ISF acts rapidly and irreversibly with lymphocytes and most likely affects events associated with the early stages of cell activation. Since immunoglobulin production represents the culmination of a complex series of inter- and intracellular events, we decided to determine the "critical" target cell through which the ISF acts. We first chose to evaluate the effect of ISF on IL-2 production, since our earlier studies suggested a role for regulatory T cells in the action of ISF on T-cell proliferation. Also, this cytokine plays a pivotal role in immunoregulatory

z

20

a

0

-1

0

18

1

21

24

TIME OF ISF ADDITION (hr)

FIG. 4. Effects of various times of exposure to ISF on immunoglobulin production. HPBL were incubated with 0.1 ng of ISF per ml for various periods before and after the addition of PWM. The cells were then incubated for 8 days after the addition of PWM, and culture supernatants were collected and assayed for IgG (A) and IgM (A) content. Results are plotted as a percentage of the amount produced in cultures receiving mitogen alone. Each point represents the mean ± SE of three experiments, each performed in triplicate. Cell viability in ISF-treated cultures was >98% of that observed in control cultures receiving mitogen alone when assessed after incubation for 8 days.

processes. As shown in Table 1, ISF did not have a significant or consistent effect on the production of biologically active IL-2. It should be noted that the dose of ISF used in these experiments exceeded the amount normally required to suppress lymphocyte responsiveness (see Fig. 2 and 3). Furthermore, addition of exogenous IL-2 to cell cultures containing ISF failed to significantly alter its inhibitory

effect. Since the function of IL-2 is in part dependent upon the expression of high-affinity IL-2R, PHA-activated cells were also assessed for the appearance of this activation marker. As shown in Fig. 5, T cells expressed IL-2R 24 h after exposure to PHA only; the addition of ISF to PHA-treated cultures failed to alter either the percentage of IL-2Rpositive cells or the surface density of IL-2R (the latter indicated by no change in the mean channel fluorescence in treated cells). We were also unable to document any increase in the release of soluble IL-2R into the culture medium; this form of the IL-2R has been implicated in impaired immune function in other systems (29). TABLE 1. Effects of ISF on IL-2 production' IL-2 (U/mi)

Culture conditions

Medium only PHA only PHA + ISF (1.25 ng) PHA + ISF (4.00 ng) PHA +ISF (20.00 ng)

Expt 1

Expt 2

0 45.6 40.4 44.2 43.7

0 41.5 30.4 32.7 33.3

a Purified T lymphocytes were incubated in the presence or absence of ISF and PHA for 24 h. The culture supernatants were harvested and assayed for IL-2 activity as described in Materials and Methods. Supernatant activities were compared with a standard containing the equivalent of 80 IU/ml.

SHENKER ET AL.

3860

INFECT. IMMUN.

L

Q-

n

E z .-

a,aL) . CD ~-4.

a), >Z

-,6 Z

CXcc.. A 0

k /

3

2

Log Fluorescence Intensity

FIG. 5. Effects of ISF on IL-2R expression by human T lymphocytes. Cells were incubated at 37°C for 24 h with PHA in the presence or absence of ISF (0.01 to 10.0 ng/ml). The cells were processed for flow cytometric analysis as described in the text. Responses to an optimal PHA (broken line) concentration were 64.3% IL-2R-positive cells with a mean fluorescence channel (MFCh) of 99. Cultures receiving both PHA and ISF (10 ng; solid line) yielded 68.0% IL-2R-positive cells with an MFCh of 101.3. Other concentrations of ISF yielded results almost identical to that observed with 10 ng of ISF. In control cultures (no PHA or ISF; dotted line), the proportion of IL-2R-positive cells never exceeded 1%. These data are representative of three separate experiments.

As an alternative approach to determining the target cell for ISF action, a series of washoff experiments were conducted in which individual cell populations were exposed to ISF and then tested for their ability to inhibit PWM-induced immunoglobulin production. As indicated above, preliminary experiments were done and demonstrated that the ISF interacted rapidly and irreversibly (within 30 min) with lymphocytes. In subsequent experiments, purified populations of T cells, B cells, and monocytes were prepared, and

each population was then exposed to ISF or medium (control) for 30 min. Following the incubation period, the cells were washed (to remove free "unbound" ISF) and placed into tissue culture with similarly treated cells or with cells not exposed to ISF. The results of these experiments are shown in Fig. 6A. When all cells were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was only observed when the B-cell-enriched population was exposed to ISF. No suppression was observed when only monocytes or T cells were treated with ISF. It should be noted that the ISF did not alter the viability of B cells when assessed 30 min (see Fig. 6 legend), 24 h, or 8 days following exposure to the inhibitory agent. Since the B-cell-enriched population contained various proportions of NK cells, we purified both populations further to determine which cell type was critical to the immunosuppressive action of ISF. Both purified subpopulations were found to be >95% pure as demonstrated by flow cytometric analysis (Fig. 7). These cells were then exposed to ISF and tested for their ability to alter immunoglobulin production. The results of these experiments (Fig. 6B) clearly indicate that the ISF functions by interacting initially with B cells. DISCUSSION Enhanced susceptibility to infectious disease is a commonly acknowledged complication in patients with overt primary and secondary immunodeficiencies. Less widely appreciated, however, are observations of immunologic dysfunction as a sequela to microbial infection in an otherwise healthy person. Furthermore, it is also becoming apparent that opportunistic organisms infecting individuals with preexisting immunologic abnormalities may contribute to the immunosuppressed status of the host. There are several mechanisms by which microbial pathogens evade or compromise the immune response of the host. Of particular interest is the production of virulence factors which may

I I

NO ISF

ALL

MONO

T-CEL B-CE31

B-CE

I

B-CW

NK CW

NW

C

1SF 1SF Cam COM CX' TYPE EXPOSED TO ISF FIG. 6. Effect of ISF on immunoglobulin production following exposure of individual cell populations. (A) Human mononuclear cells were fractionated into three populations, monocytes (MONO), T cells, and B cells. Each population was then exposed to ISF (1 ng/ml) or medium for 30 min, washed, and placed into culture with PWM. Culture supernatants were collected and assayed for IgG (solid bars) and IgM (hatched bars) levels by ELISA. The results are plotted as a percentage of the amount produced by control cultures in which all cells were exposed to medium. Each bar represents the mean + SE of three experiments, each performed in triplicate. (B) The B-cell-enriched population was further fractionated into a population of pure B cells and another population of pure NK cells. Both cell types were treated and cultured as described above; in these studies, all cultures received T cells and monocytes that were exposed to medium only. Cell viability was also assessed following exposure to ISF and averaged 97, 98, and 98% for T cells, B cells, and monocytes, respectively. CGM, Complete growth medium.

IMMUNE SUPPRESSION BY A. ACTINOMYCETEMCOMITANS

VOL. 58, 1990

Log Fluorescence Intensity

FIG. 7. Flow cytometric analysis of purified B and NK cells. (A) Purified B cells were labeled with anti-CD20 monoclonal antibody conjugated to fluorescein isothiocyanate and analyzed by flow cytometry. Solid line, Labeled cells; broken line, isotype control. (B) Purified NK cells were labeled with anti-CD16 conjugated to fluorescein isothiocyanate. Solid line, labeled cells; broken line, isotype controls.

aggressively subvert the immune response (reviewed in references 31 and 44). Such microbial products represent an important source of immunoregulatory agents which may affect the immune response via several different mechanisms. For example, cholera toxin has been shown to interact with lymphocytes both in vitro and in vivo and to activate adenylate cyclase, resulting in the intracellular accumulation of cyclic AMP (12, 40). On the other hand, products of Corynebacterium parvum, Treponema denticola, and T. pallidum affect macrophages, provoking the release of mediators capable of inhibiting lymphocyte function (21, 36, 41). T-regulatory cells are also a potent target for microbial modulation of the immune response (7, 20). Other microorganisms, such as Trypanosoma cruzi, act by interfering with IL-2R expression (2). We have previously shown that A. actinomycetemcomitans produces a protein capable of inhibiting DNA, RNA, and protein synthesis in human T cells activated by mitogens or antigens (33, 38). In this report, we have demonstrated that the purified ISF was capable of inhibiting IgG and IgM production by human B cells in response to PWM. As we had observed with T-cell proliferation, the effects on immunoglobulin production were dose dependent. It was clear from these studies that immunoglobulin production was significantly more sensitive to the suppressive effects of the ISF than were the proliferative responses of T cells. The ISF appeared to affect immunoglobulin production by interfering with the early stages of cell activation. While our earlier observations on T-cell proliferation indicated that the ISF interacted rapidly and irreversibly with lymphocytes, it was clear that inhibition was not manifest until 48 to 72 h after exposure (38). We had demonstrated that this early requirement for the ISF followed by the delay in reduced DNA synthesis might be associated with the generation of suppressor T cells. In fact, we have reported previously on the activation and expansion of a subpopulation of suppressor T cells 48 to 96 h following exposure to ISF (33). Our current observations also suggest that the ISF interacts rapidly with lymphocytes, but in contrast to our earlier findings, it is evident that the initial interaction is with the B cell. It is not clear at this time whether these discrepancies

3861

reflect differences in the direct effects of the ISF on T-cell proliferation as opposed to its effect on immunoglobulin production by B cells. Alternatively, it is feasible that suppression of B-cell and T-cell function is mediated by a similar and interrelated mode of action involving immunoregulatory cells that are capable of modulating the function of both cell types. Clearly, T-cell proliferation and PWMinduced immunoglobulin production have been shown to be subject to the regulatory influences of T-helper and T-suppressor cells as well as monocytes. Thus, it is entirely feasible to propose that the ISF acts initially with B cells, which in turn function in a regulatory role. In this regard, there have been numerous reports of an immunoregulatory function(s) associated with B-cell subpopulations. These include activities associated with antigen presentation, production of anti-idiotypic antibodies, and, more recently, suppressor cell activity (5, 6, 9, 10, 15, 17, 18, 23, 42). Such cellular interactions could ultimately result in direct suppression via a B-suppressor cell or the induction of other immunoregulatory pathways involving T-suppressor cells. In summary, recent studies suggest a strong association between A. actinomycetemcomitans infection and the etiology of juvenile periodontitis (reviewed in reference 46). Although the immunologic mechanism(s) involved in periodontal disease is not clearly defined, there is substantial evidence that impaired host defense mechanisms may contribute to the disease process. A. actinomycetemcomitans, in particular, may be able to upset antibacterial defense mechanisms in the gingival crevice. While we do not know the exact mechanism by which the A. actinomycetemcomitans ISF acts to cause immunosuppression, it would appear to involve effects on both B cells and T-regulatory cells. Hence, bacterium-derived immunosuppressive factors may contribute to the pathogenesis of infectious disease in general and periodontal disease in particular. Such factors could lead to a state of hyporesponsiveness that favors colonization by the initiating organism or by other opportunistic

organisms. ACKNOWLEDGMENTS We are grateful to Drs. Malamud, Golub, and Lally for their advice and helpful discussions in protein purification and characterization, Elizabeth Ruggieri for her technical assistance, and Alan Pickard for flow cytometric analysis. This work was supported by Public Health Service grants DE06014, DE00170, DE08587, and DE07118 from the National Institutes of Health.

LITERATURE CITED 1. Arneborn, P., and G. Biberfeld. 1983. T-lymphocyte subpopulations in relation to immunosuppression in measles and varicella. Infect. Immun. 39:29-37. 2. Beltz, L. A., F. Kierszenbaum, and M. B. Sztein. 1989. Selective suppressive effects on Trypanosoma cruzi on activated human lymphocytes. Infect. Immun. 57:2301-2305. 3. Boyum, A. 1968. Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21:77-89. 4. Bromley, G. S., and M. Solender. 1986. Hand infection caused by Actinobacillus actinomycetemcomitans. J. Hand Surg. llA: 434-436. 5. Cuff, C. F., B. Packer, V. Rivas, C. M. Rogers, A. Cassone, R. Donnelly, and T. J. Rogers. 1988. Induction of immunosuppressive B-lymphocytes with components of Candida albicans. Adv. Exp. Med. Biol. 239:367-378. 6. Farkas, R., Y. Manor, and A. Klajman. 1986. Generation of B suppressor cells by phytohaemagglutinin. Immunology 57:395398. 7. Fidel, P. L., and J. W. Murphy. 1988. Characterization of an in

3862

8.

9. 10. 11. 12.

13.

14.

15.

16.

SHENKER ET AL.

vitro-stimulated, Cryptococcus neoformans-specific second-order suppressor T cell and its precursor. Infect. Immun. 56:12671272. Fulwyler, M. J., C. W. McDonald, and J. L. Haynes. 1979. The Becton Dickinson cell sorter, p. 653-667. In M. Melamed, P. Mullany, and M. Mendelsohn (ed.), Flow cytometry and cell sorting. John Wiley & Sons, Inc., New York. Gilbert, K. M., and M. K. Hoffmann. 1983. Suppressor B lymphocytes. Immunol. Today 46:545-547. Hausman, P. B., D. H. Sherr, and M. E. Dorf. 1986. Antiidiotypic B cells are required for the induction of suppressor T cells. J. Immunol. 136:48-53. Hofstad, T., and A. Stallemo. 1981. Subacute bacterial endocarditis due to Actinobacillus actinomycetemcomitans. Scand. J. Infect. Dis. 13:78-79. Holmgren, J., and L. Lindholm. 1977. Cholera toxin, ganglioside receptors and the immune response, p. 77-96. In B. Cinader (ed.), Immunology of receptors, immunology series. Marcel Dekker, Inc., New York. Hunt, S. V. 1986. Preparative immunoselection of lymphocyte populations, p. 55.1-55.18. In D. M. Weir (ed.), Handbook of experimental immunology, vol. 2. Blackwell Scientific Publications, Boston. Kaplan, G., R. R. Gandhi, D. E. Weinstein, W. R. Levis, M. E. Patarroyo, P. J. Brennan, and Z. A. Cohn. 1987. Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro. J. Immunol. 138:3028-3034. Kunicka, J., and C. D. Platsoucas. 1988. Leukaemic B cells from patients with chronic lymphocytic leukaemia suppress immunoglobulin production by lymphocytes from normal donors. Scand. J. Immunol. 28:1-10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)

INFECT. IMMUN.

27.

28. 29.

30.

31. 32. 33. 34.

35.

36.

227:680-685. 17. L'age-Stehr, J., H. Teichmann, R. K. Gershon, and H. Cantor. 1980. Stimulation of regulatory T cell circuits by immunoglobulin dependent structures on activated B cells. Eur. J. Immunol. 10:21-26. 18. Lakey, E. K., L. A. Casten, W. L. Niebling, E. Margoliash, and S. K. Pierce. 1988. Time dependence of B cell processing and presentation of peptide and native protein antigens. J. Immunol. 140:3309-3314 19. Lombardi, G., D. Vismara, E. Piccolella, V. Colizzi, and G. L. Asherson. 1985. A non-specific inhibitor produced by Candida albicans-activated T cells impairs cell proliferation by inhibiting interleukin-1 production. Clin. Exp. Immunol. 60:303-310. 20. Markham, R. B., G. B. Pier, and W. G. Powderly. 1988. Suppressor T cells regulating the cell-mediated immune response to Pseudomonas aeruginosa can be generated by immunization with antibacterial T cells. J. Immunol. 141:3975-3979. 21. Metzger, Z., J. T. Hoffeld, and J. J. Oppenheim. 1980. Macrophage mediated suppression. I. Evidence for participation of both hydrogen peroxide and prostaglandins in suppression of murine lymphocyte proliferation. J. Immunol. 124:983-988. 22. Olson, G. B., P. B. Dent, W. E. Rawls, M. A. South, J. R. Montgomery, J. L. Melnick, and R. A. Good. 1968. Abnormalities of in vitro lymphocyte responses during rubella virus infections. J. Exp. Med. 128:47-68. 23. Paglieroni, T., V. Caggiano, and M. Mackenzie. 1988. CD5 positive immunoregulatory B cell subsets. Am. J. Hematol. 28:276-278. 24. Parra, C., L. F. Montano, M. Huesca, I. Rayon, K. Willms, and F. Goodsaid. 1986. Inhibition of mitogenesis induced by phytohemagglutinin and Lens culinaris lectin in adherent-cell supernatants treated with protein extract of Mycobacterium tuberculosis. Infect. Immun. 52:309-313. 25. Patel, P. K., and M. W. Seitchik. 1986. Actinobacillus actinomycetemcomitans: a new cause for granuloma of the parotid gland and buccal space. Plastic Reconstructive Surg. 77:476478. 26. Reed, S. G., D. L. PihI, and K. H. Grabstein. 1989. Immune deficiency in chronic Trypanosoma cruzi infection. Recombi-

37.

38.

39. 40.

41.

42.

43.

44. 45.

nant IL-1 restores Th function for antibody production. J. Immunol. 142:2067-2071. Reiner, N. E. 1987. Parasite accessory cell interactions in murine leishmaniasis. I. Evasion and stimulus-dependent suppression of the macrophage interleukin 1 response by Leishmania donovani. J. Immunol. 138:1919-1925. Rivas, V., and T. J. Rogers. 1983. Studies on the cellular nature of Candida albicans-induced suppression. J. Immunol. 130:376379. Rubin, L. A., C. C. Kurman, M. E. Fritz, W. E. Biddison, B. Boutin, R. Yarchoan, and D. L. Nelson. 1985. Soluble interleukin-2 receptors are released from activated human lympoid cells in vitro. J. Immunol. 135:3172-3177. Salman, R. A., S. J. Bonk, D. G. Salman, and R. S. Glickman. 1986. Submandibular space abscess due to Actinobacillus actinomycetemcomitans. J. Oral Maxillofac. Surg. 44:1002-1005. Schwab, J. H. 1975. Suppression of the immune response by microorganisms. Bacteriol. Rev. 39:121-143. Shenker, B. J. 1987. Immunologic dysfunction in the pathogenesis of periodontal diseases. J. Clin. Periodontol. 14:489-498. Shenker, B. J. 1988. Immunosuppression: an etiopathogenic mechanism, p. 178-186. In B. Guggenheim (ed.), Periodontology today. S. Karger AG, Basel. Shenker, B. J., P. Berthold, P. Dougherty, and K. K. Porter. 1987. Immunosuppressive effects of Centipeda periodontii: selective cytotoxicity for lymphocytes and monocytes. Infect. Immun. 55:2332-2340. Shenker, B. J., and J. M. DiRienzo. 1984. Suppression of human peripheral blood lymphocytes by Fusobacterium nucleatum. J. Immunol. 132:2357-2362. Shenker, B. J., M. A. Listgarten, and N. S. Taichman. 1984. Suppression of human lymphocyte responses by oral spirochetes: a monocyte-dependent phenomenon. J. Immunol. 132: 2039-2045. Shenker, B. J., and W. Matt. 1987. Suppression of human lymphocyte responsiveness by forskolin: reversal by 12-0tetradecanoyl phorbol-13-acetate, diacylglycerol and ionomycin. Immunopharmacology 13:73-86. Shenker, B. J., W. P. McArthur, and C. C. Tsai. 1982. Immune suppression induced by Actinobacillus actinomycetemcomitans. I. Effects on human peripheral blood lymphocyte responses to mitogens and antigens. J. Immunol. 128:148-154. Shenker, B. J., and J. Slots. 1989. Immunomodulatory effects of Bacteriodes products on in vitro human lymphocyte functions. Oral Microbiol. Immunol. 4:24-29. Shenker, B. J., W. F. Willoughby, M. R. Hollingdale, and T. N. Mann. 1980. In vivo responses to inhaled proteins. III. Inhibition of experimental immune complex pneumonitis after suppression of peripheral blood lymphocytes. J. Immunol. 124: 1763-1772. Tomai, M. A., B. J. Elmquist, S. M. Warmka, and T. J. Fitzgerald. 1989. Macrophage-mediated suppression of Con A-induced IL-2 production in spleen cells from syphilitic rabbits. J. Immunol. 143:309-314. Tsukuda, K., Y. Tsukuda, and G. Klein. 1981. Suppressor T cells activated by lymphoblastoid cell lines inhibit pokeweed mitogen-induced immunoglobulin synthesis. Cell. Immunol. 60: 191-202. Wahl, L. M., I. M. Katona, R. L. Wilder, C. C. Winter, B. Haraoui, I. Scher, and S. M. Wahl. 1984. Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation. I. Characterization of B-lymphocyte, T-lymphocyte and monocyte-enriched fractions by flow cytometric analysis. Cell. Immunol. 85:373-383. Weir, D. M., and C. C. Blackwell. 1983. Interaction of bacteria with the immune system. J. Clin. Lab. Immunol. 10:1-12. Wysocki, L., and V. Sato. 1978. Panning for lymphocytes: a method for cell separation. Proc. Natl. Acad. Sci. USA 75:

2844-2848. 46. Zambon, J. J. 1985. Actinobacillus actinomycetemcomitans in human periodontal disease. J. Clin. Periodontol. 12:1-20.