CELLULAR IMMUNOLOGY 127,327-336 (1990)

Action of Pertussigen (Pertussis Toxin) on Serum IgE and on FCC Receptors on Lymphocytes

JOHN J. MUNOZ AND MARIUS G. PEACOCK

Department QfHealth and Human Services, Public Health Service, National Institutes of Health, National Institute ofAllergy and Iqfectious Diseases, Laboratory of Vectors and Pathogens,

Rocky Mountain Laboratories, Hamilton, Montana 59840

Received December I I 1989; accepted January 9, 1990

Pertussigen (pertussis toxin (PT)) is one of the most effective stimulators of IgE production in mice and rats. Employing flow microfluorimetric analysis (FMF), we showed that PT increases the percentage of blood and spleen lymphocytes with IgE on their surface. The percentage of IgE-bearing cells in the spleen of normal untreated C57Bl/lOSCN mice of various ages varied from 2.2 to 12.2%, with an average value of 6.1 + 5.4%. In mice treated with 400 ng of PT and 1 mg of chicken egg albumin (EA), the percentage of these cells increased, 14 days after immunization, to an average value of 3 I. 1 * 2.2%. Immunization of mice with PT alone in- crease the percentage of IgE-bearing cells only slightly (13. I -t 2.2% of the splenic lymphocytes) while injection of 1 mg of EA alone did not have any detectable action. As little as 6 ng of PT, when given simultaneously with I mg of EA, increased the percentage of IgE-bearing lympho- cytes. A booster dose of 10 rg of EA given on Day I4 induced a further increase in the percentage ofthese cells even when as little as 0.039 ng of PT had been given at the time of initial immuniza- tion. PT was effective when given 4 days before or 5 days after EA. EA was effective when given 4 days before or 4 days after PT, but not 8 days after. The increase in IgE-bearing cells was mainly due to cytophilic binding of IgE to receptors for the c chain of IgE (Fee) on the surface of lymphocytes rather than to a greater number of IgE-producing cells. This was shown by remov- ing the IgE from FCC receptors by acid treatment which reduced the percentage of IgE-bearing cells to nearly normal values. The antibodies of IgE class with specificity to EA were increased dramatically, while antibodies with specificity to PT were not detected. o 1990 Academic PKSS, IX.

INTRODUCTION

The stimulation by pertussigen (PT) of immunoglobulin E (IgE) with specificity to the antigen given is well known (17). As early as 1964, Mota (13-15) showed that pertussis vaccine stimulates the production of “mast-cell-sensitizing antibodies,” now known to be IgE. We (2, 19) showed that PT is the active substance in the Borde- tellu pertussis cell responsible for stimulation of IgE production and that as little as 0.1 ng of this toxin is effective ( 16). Ishizaka et al. (7) have shown that PT stimulates a subset of lymphocytes to produce a glycosylating enzyme which converts an IgE- binding factor into a stimulator of IgE production. Some (23,24) think that PT stimu- lates IgE production by decreasing suppressor cells. The increase of IgE induced by PT could be due to an increased production per cell or to proliferation of cells produc- ing IgE. We wanted to find the extent of this increase at the cellular level. Thus, we studied the action of PT on the percentage of lymphocytes expressing IgE on their

327

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328 MUNOZ AND PEACOCK

surface by means of flow microfluorimetric analysis (FMF). A marked increase of @-bearing cells was observed in mice receiving PT and chicken egg albumin (EA), but, as found by Katona et al. (8) in mice infected with Nippostrongylus brasiliensis, this was mainly due to an increase in the cytophilic binding of IgE to IgE receptors on lymphocytes, rather than to a proliferation of IgE-producing cells. The results of these investigations are described in this paper.

MATERIALS AND METHODS

Mice. Female C57Bl/ 1OSCN were raised in our animal facilities. The ages varied from 4 to 10 weeks. Within each experiment, the experimental and control mice were of the same age.

Antisera. For the FMF analysis we employed the following antisera: (i) A rabbit antiserum specific to the E chain of mouse IgE, which was kindly supplied to us by Dr. Alfred Nisonoe (ii) fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit Ig purchased from Cappel Laboratories (Downingtown, PA); and (iii) a FITC-conju- gated goat anti-mouse Ig with specificity to mouse IgG, , IgG2,, IgGlb, and IgG3 pur- chased from Becton-Dickinson (Mountain View, CA).

For the enzyme-linked immunosorbent assay (ELISA) tests the following antisera were used: (i) A rat monoclonal antibody specific to the E chain of mouse IgE, pur- chased from United States Biochemical Corp. (Cleveland, OH), and (ii) an alkaline phosphatase-labeled rabbit anti-rat IgG + IgM (H + L chain) purchased from Zymed Laboratories, Incorporated (South San Francisco, CA).

A FITC-conjugated goat anti-mouse serum purchased from Becton-Dickinson was used for the detection of immunoglobulin-bearing cells.

Pertussigen. PT was purified by the method of Sekura et al. (20), except that it was not precipitated with ammonium sulfate and instead was dialyzed extensively against water, lyophilized, and kept in a desiccator under vacuum at 2-5°C. When needed, it was weighed and dissolved in 0.08 M DL-cu-alanine made in 0.0 15 M formic acid, pH 3.44. Immediately after PT was dissolved, the solution was diluted with an equal volume of 0.05 M Tris buffer made in 1 A4 sodium chloride, pH 8. At a final concen- tration of 1 mg per milliliter, the solution was distributed in 30-~1 volumes into small plastic tubes, capped, and placed in a sealed plastic bag at -20°C. For each experi- ment, the content of one tube was diluted with physiological saline to give the desired concentration.

Antigens. The antigen used in most experiments was crystalline chicken egg albu- min (EA) purchased from Sigma Chemical Co. (St. Louis, MO). Keyhole limpet he- mocyanin (KLH) purchased from Calbiochem (San Diego, CA) and a dinitrophenol- conjugated bovine serum albumin made in our laboratory were also used in one experiment. Solutions of these antigens were made in physiological saline. IgE was obtained by ammonium sulfate precipitation from hybridoma fluid of cultured TIB- 14 1 cells purchased from American Type Culture Collection (Rockville, MD).

Preparation of cells andflow microfluorimetric analysis. Mice were bled from the ophthalmic venous plexus and killed by cervical dislocation. The spleens were re- moved and placed in Dulbecco physiological buffered saline containing 2% fetal calf serum (FCS) and 0.1% sodium azide (DPBS), and single-cell suspensions were made by standard techniques (5). The blood leukocytes were separated by the Ficol-Hy- paque centrifugation method (5) and then treated as the spleen cell suspensions. The

ACTION OF PERTUSSIGEN ON IgE AND FCC RECEPTORS 329

suspension of spleen cells was adjusted to 10’ cells per milliliter, while the suspension of blood leukocytes was only 106. Before the final suspensions were made, the cells were centrifuged through a 4-ml layer of FCS at 2008 for 5 mitt, followed by passage through nylon mesh (74-pm mesh opening, Small Parts, Miami, FL). One-hundred microliters of the spleen or blood cell suspensions were incubated for 15 min with 100 ~1 of the appropriate antibody diluted to a predetermined optimal concentration. The cells were washed with 200 ~1 of DPBS. When cells were treated with nonfluo- resceinated rabbit anti-mouse, a further treatment with FITC-conjugated goat anti- rabbit immunoglobulin was done. After being washed, the cells were analyzed with the fluorescence activated-cell sorter (FACS analyzer from Becton-Dickinson, Mountain View, CA). The analyzer was operated in a log gain mode with full-scale fluorescence being 3.0 log. Volume gates were set to exclude cells smaller and larger than lymphocytes. For the estimation of the percentage of the different cell types in the cell suspensions, the FACS analyzer was gated to record at least 99.5% of the unstained leukocytes as negative for fluorescence. Those cells containing the fluores- ceinated antibody were recorded as a percentage of the 10,000 cells counted. In most experiments, four mice per group were employed.

Acid treatment of cells to remove IgEfrom FCC receptors. It is known that the IgG, IgA, and IgE attached to cell receptors can be removed by acid treatment of the cells (8, 10). In this work, the method of Kumagai et al. ( 10) as described by Katona et al. (8) was used. Briefly, 0.5 ml (about 10’ cells) of a spleen-cell suspension, collected and processed as for FMF analysis were sedimented by centrifugation at 400g for 5 min, the supernatant was removed, and 1 ml of 0.05 Macetate buffer (pH 4), contain- ing 0.085 M sodium chloride, 0.005 M potassium chloride, and 1% FCS was added. The resuspended cells were kept in an ice bath for 1 min, and then 10 ml of 0.1 M Tris (pH 7.4) containing 1% FCS was added to neutralize the acid to pH 7.3-7.4. The cells were sedimented at 400g for 10 min, washed twice with 5-ml portions of DPBS. The cells were then resuspended in 1 ml of DPBS filtered through a 74-pm nylon mesh, stained with anti-IgE, washed, and treated with fluoresceinated goat anti- mouse globulin. The percentage of IgE-bearing cells was determined by FMF analysis as described above.

Antibody determinations. Antibodies of IgE isotype against EA were assayed by ELISA tests performed in 96-well plastic microtiter plates (Limbro/Titertek plates purchased from Flow Laboratories, Inc., McLean, VA), coated with EA by standard techniques (4) and also by passive cutaneous anaphylaxis (PCA) tests as previously described (2, 18).

RESULTS

Eflect of PT on the percentage of lymphocytes expressing IgE on their surface. C57Bl/lOSCN mice in groups of four were immunized with 1 mg of EA was given intraperitoneally (ip) and 400 ng of PT was given intravenously (iv). Fourteen days later, the mice were bled, and splenic leukocyte suspensions made from each speci- men. From each of four normal mice of the same age and sex, similar cell suspensions were made. The cell suspensions were treated with either anti-Ig or anti-IgE, and the percentage of each cell type was determined by the FMF analysis. Figure 1 shows that treatment with 400 ng of PT and 1 mg of EA markedly increased the percentage of IgE-bearing cells in the spleen (Figs. 1 and 2). This increase in IgE-bearing cells was also present in the blood leukocytes (Fig. 3).

330

Untreated cells

MUNOZ AND PEACOCK

Treated with anti-lg

t., B IBB 286

FL,

Treated with anti-IgE

1 FL,

FIG. 1. Histograms obtained with spleen cells from normal (A, B, C) and 400-ng-PT- plus I-mg-EA- treated (D, E, F) mice 14 days after treatment. Fluorescence intensity is in the abscissa and number of events in the ordinate. The cells not containing the FITC-conjugated antibody are on the left side of the gate placed at FL-I 80. The number ofcells counted per sample was 10,000.

The effect of different doses of PT on the percentage of IgG-bearing lymphocytes (Fig. 4) and on the percentage of IgE-bearing lymphocytes (Fig. 5) in spleen cell sus- pensions was determined 14 days after PT and EA, and 7 days after a booster of 10

50

40

"p 5 B Z 30 a c

i "a 20

H 10

Treatment: 400 ng Ptx IV +

1mgEaIP + z + -

FIG. 2. Effect of 400 ng PT on the percentage of spleen cells bearing IgE. Four mice per group were employed. The graph gives the average -C SD of the percentage of lymphocytes bearing IgE on their surface. (+) means treatment and (-) no treatment with the corresponding substance.

ACTION OF PERTUSSIGEN ON IgE AND Fee RECEPTORS 331

Treatment: 400 ng Ptx IV + -

1 mg EalP + -

FIG. 3. Effect of 400 ng PT on the percentage of blood leukocytes bearing IgE. Four mice per group were employed. The graph gives the average rt_ SD of the percentage of lymphocytes bearing IgE on their surface. (+) means treatment and (-), no treatment with the corresponding substance.

pg EA given on Day 2 1. It is evident that 14 days after injection of 0.39 to 25 ng of PT and 1 mg EA, no striking effect on the percentage of IgG-bearing cells was noticed. However, at PT doses of 100 and 400 ng, the percentage of these cells was lower than the values obtained with mice receiving only EA (Fig. 4) or with unprimed mice (57.37 + 3.32) probably due to the marked increase in T-cells by these high doses of PT. On Day 28 (7 days after a booster dose of EA), the only dose of PT giving a decrease in the percentage of IgG-bearing cells was 400 ng (only one mouse was avail- able in this group). The results in Fig. 5 show strikingly different results when the percentage of IgE-bearing cells was determined after a single dose of PT and EA. A dose-dependent increase in IgE-bearing cells was observed from a dose of 6.25 ng to 400 ng of PT (larger doses of PT are toxic). After a booster dose of EA, as little as 0.39 ng of PT gave a detectable increase in the percentage of IgE-bearing cells, and 1.56 ng gave an optimal response.

Other antigens such as KLH and DNP-BSA could be substituted for EA with sim- ilar increases in the percentage of IgE-bearing cells (data not shown).

Optimal time to administer PT and EA to increase the IgE-bearing lymphocytes in the spleen. The time at which 400 ng of PT were administered with respect to the injection of 1 mg of EA was varied from 4 days before to 5 days after. All injections of PT were given iv, and the EA was given ip. The spleens were removed 14 days after the injection of EA. PT was effective in increasing the IgE-bearing cells in the spleen when given 4 days before EA or 5 days after, but the optimal time was from 3 days before to 1 day after EA (Fig. 6).

In another experiment, the time at which EA was given with respect to PT was varied. The spleens were removed 14 days after the PT injection. EA was given up to 4 days before and 8 days after the PT injections. The percentages of IgE-bearing cells determined by FMF were 23 +- 5.9 and 30.6 ? 2.5 when EA was given 2 or 4 days

332 MUNOZ AND PEACOCK

60 -

= Pmboost

El q Poaboosl

0 39 1.56 6.25 25 100 400

IV dose of Ptx in nanograms

FIG. 4. Effect of treatment with different doses of PT plus 1 mg of EA on the percentage of spleen cells bearing IgG. Three or four mice per group were used. The postboost groups received 10 pg of EA ip on Day 2 1 and the spleens were collected 7 days later.

before PT, respectively, and 24.5 f 6.4, 25.2 + 4.4, 31.9 + 1.3, and 13.4 f 3.4 when given simultaneously, 2, 4, or 8 days after PT, respectively. The control values in mice receiving PT alone were 15.7 + 6.0 and in unprimed mice 13.4 f 5.4. Thus, the action of PT in increasing IgE-bearing cells was detected when EA was given 4 days before to 4 days after PT, but not when given 8 days after. This last group of mice had been exposed to EA for only 7 days before the spleens were removed, and thus may have not had sufficient time to show the response.

Eflect of acid treatment on the percentage of IgE-bearing cells. Six-week-old female mice were immunized with 1 mg EA given ip and 200 ng of PT given iv. Fourteen days later, the spleens were collected to analyze the cells for the presence of IgE. Half of the cell suspension was treated with acetate buffer, pH 4, as described under Materials and Methods, and the other half was left untreated. Both preparations were analyzed for the presence of IgE and IgG-bearing cells by FMF analysis. There was no significant drop in the percentage of IgG-bearing cells by acid treatment (data not shown), but, as seen in Fig. 7, the percentage of IgE-bearing cells dropped from 27.1 f 1.13 to 8.5 + 0.99 in the spleen cells of mice receiving PT + EA. This corresponded to a drop of nearly 70%. The values after acid treatment were still slightly elevated compared to those obtained from PT-treated, EA-treated, or unprimed mice indicat-

ACTION OF PERTUSSIGEN ON IgE AND FCC RECEPTORS 333

0.0 0.39 1.56 6.25 25 100 400

IV dose of Ptx in nanograms

FIG. 5. Effect of treatment with different doses of PT plus I mg of EA on the percentage of spleen cells bearing IgE. Three or four mice per group were used. The postboost groups received 10 pg of EA ip 2 1 days after the initial immunization, and the spleens were collected 7 days later.

ing an increase in IgE-producing cells. These results have been duplicated repeatedly with PT + EA and also with EA and various whole cell B. pertussis vaccines made from strains that produce PT. After the IgE was removed from the low-affinity Fct

f 10 0.

-4 -3 -2 0 +l l 3 +5

Day of Injactii of F’bt with reference lo adminiitratii of Ea

I!

FIG. 6. Effect of time of treatment with 400 ng PT on the percentage of spleen cells bearing IgE. Three or four mice per group were used.

334 MUNOZ AND PEACOCK

= untreated cells

la = acid-treated cells

Treatment: 100ngPtxIV + +

1 mg Ea IP + +

FIG. 7. Effect ofstripping IgE from FCC receptors by acetate buffer (pH 4) treatment ofspleen lymphocytes on the percentage ofcells bearing IgE. Three or four mice were used per group.

receptors, IgE could be reabsorbed to the receptor again by treating the cells with fluids from IgE-producing hybridoma culture. This process was repeated two succes- sive times with the same cell suspension with similar results (data not given).

Stimulation of IgE antibody response to EA by PT. PT is known to increase the antibody response to protein antigens ( 16) and a booster dose of the antigen increases further the response (17, 19). This activity was also observed in the present work employing the C57Bl/lOSCN mice (Table 1). When antibodies were measured by

TABLE 1

Effect of PT on the Antibody of IgE Isotype with Specificity to EA”

Anti-EA by ELISA Anti-EA by PCA

14 days 28 daysb 14 days 28 daysb

373 5120 200 500 853 5120 50 500 133 5120 50 <500 133 5120 &50 500 133 3200 <50 100 67 480 <50 t20

<20 <20 <50 <20

LI C57BI/lOSCN mice were given PT iv and, at the same time, 1 mg EA ip. Fourteen days later, the mice were bled. On Day 21, the mice received an ip booster dose of 10 rg of EA. On Day 28, they were bled again.

b Titers 7 days after booster. Values given are the reciprocal of the serum dilution still giving a positive test. In the ELISA test a positive reaction was an OD of 0.2 or greater, and for the PCA a blue spot on the skin site of at least 5 mm in diameter.

ACTION OF PERTUSSIGEN ON IgE AND FCC RECEPTORS 335

ELISA at Day 14, and 7 days after the booster dose of EA, the smallest dose of PT that significantly increased the IgE titer of antibodies to EA was less than 0.39 ng (the smallest dose tested). When antibodies to EA were measured by PCA, 6.25 ng or greater doses of PT increased the 14-day preboost titers, and 1.56 ng or greater in- creased the postboost titer. Due to the marked differences in the sensitivity of the two tests, and to the variability of the in viva assays, these two titrations do not show a perfect correlation.

DISCUSSION

PT is one of the best known stimulators of IgE in mice and rats (2, 13- 17, 19). This action can be induced with as little as 0.1 ng given at the time of immunization with a protein antigen, and the titers of antibody of the IgE isotype specific to the antigen given are increased over 100 times those in sera of mice receiving the antigen alone ( 16, 19, and present work). When EA and PT are given to C57Bl/ 1OSCN mice, a marked increase in the lymphocytes expressing IgE on their surface was observed in the spleen, blood, and lymph node cells (data for lymph node cells not given). When the cells were treated with acetate buffer at pH 4, most of the surface IgE was stripped off, indicating that the IgE was bound to low-affinity FCC receptor (8, 1 I), rather than to cells producing IgE. Interestingly, similar observations have been made in mice infected with Nippostrongylus brasiliensis (8), an infection which also stimulates the production of IgE. The percentage of lymphocytes with Fct receptors is also increased with age of mice (26). In our hands, lo- to 14-week-old mice also had a greater per- centage of IgE-bearing cells than did 4- to 6-week-old mice (data not given). The effect of treatment with PT and EA, however, was demonstrated in young as well as older mice, but the percentages of IgE-bearing cells were higher in the latter group. It is important to emphasize that PT alone at the doses given (up to 400 ng) increased the percentage of IgE-bearing cells only slightly and 1 mg EA alone did not have any effect on these cells. The mechanism by which the increase in the percentage of cells with Fct receptors occurs is not yet clear. In our experiment, this increase occurred when IgE antibodies to EA were also increased. Furthermore, the requirement of an antigen to induce this marked increase may indicate that the stimulation of IgE production to an antigen may play an important role in the increase of Fee receptors. In fact, it has already been demonstrated that IgE increases FCC receptors (12). Ac- cording to Ishizaka et al. (7), PT plus an antigen increase IgE by the production of IgE- binding factors by macrophages and by increasing the production of a glycosylating enzyme by a subset of T-lymphocytes. These two actions of PT result in the glycosyla- tion of IgE-binding factors which in this form become stimulators of IgE production by plasma cells. This action of PT requires the presence of an intact S 1 subunit of the PT molecule since mutant strains of B. pertussis that produce PT with a modified S 1 do not induce immunopotentiating action (1). It has also been found that these B. pertussis mutants failed to induce an increase in the binding of IgE to receptors in lymphocytes (unpublished work). These observations again support the view that the stimulation of IgE production may be responsible for the augmentation of the cytophilic binding of IgE to lymphocytes.

Recently, it has been shown that IL-4 is a lymphokine that stimulates IgE produc- tion (3) and also FCC receptors in B cells (6,9). It is likely that PT may act by stimulat- ing the production of this lymphokine, although the production of other lymphokines

336 MUNOZ AND PEACOCK

are also affected by PT (2 1,22). The work presented clearly shows that PT increases IgE with specificity to the antigen (EA) given and FCC receptors in lymphocytes which from the work of others (25,26) should be mainly B-lymphocytes.

ACKNOWLEDGMENTS

We express our thanks to Drs. Seth Pincus and Richard Buller for their suggestions for improving the manuscript, to Betty Kester for typing the manuscript, and to Robert Evans and Gary Hettrick for making all the illustrations. We also thank Dr. Alfred Nisonoff for supplying us with a specific anti-mouse IgE antibody.

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