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Differential effects of pharmacologic agents on EAC3 rosette formation by lymphocytes from normal and asthmatic subjects
Differential effects of pharmacologic agents on EAC3 rosette formation by lymphocytes from normal and asthmatic subjects Barry A. Kohn, M.D., Major, United States Air Force, Medical Corps, Frank J. Twarog, M.D., Ph.D.,* and Raif S. Geha, M.D. Boston, Mass.
We studied the effect of isoproterenol hydrochloride (I) and theophylline (Th) on EAC3 rosette formation by lymphocytes from 30 normal adult subjects, 35 asthmatic subjects, 11 subjects who were age and sex matched with the asthma group, and 10 subjects with allergic rhinitis. Baseline EAC3 rosette numbers were similar for all groups (normal adults, 13.2%; normal children, 15.190; rhinitis, 14.490; asthma, 14.6%). Incubation with I and Th in the presence of fetal calf serum caused enchancement of EAC3 rosette formation by lymphocytes from normal adults (mean increase, 52% with I and 53% with Th), from normal age- and sex-matched controls (mean increase, 45% with I and 4090 with Th), f rom subjects with allergic rhinitis (mean increase, 50% with I and 52% with Th), but not by lymphocytes from asthmatic subjects (mean increase, 0% with I and 290 with Th). These results were not affected by bronchodilator therapy. Similar results were obtained with cholera toxin, prostaglandin E,, and adrenaline, suggesting that the defect in lymphocytes from asthmatic patients resides at the level of 3’, 5’qclic adenosine monophosphate (CAMP) production or at the level of CAMP-triggered events. Lymphocyte fractionation experiments into T cell-rich, B cell-rich, and null cell populations revealed that the null cell population was a target for the l-mediated enhancement of EAC3 rosette formation. Null cells from normal subjects, but not from asthmatic subjects, exhibited an increase in the number of EAC3 rosettes formed following incubation with I. This enhancement required protein synthesis as it was prevented by the addition of puromycin hydrochloride. The resistance of peripheral blood lymphocytes from asthmatics to the enhancing effect of I and Th on EAC3 rosette formation may be used to study the biochemical defect in asthma and should be assessed for possible use in the detection of the latent asthmatic.
Beta adrenergic blockade was first postulated by Szentivanyi 1 to explain the defect in bronchial asthma. Subsequent studies have suggested that asthmatics have altered metabolic and cellular responses to isoproterenol. *-l* The adrenergic effects of isopro-
From the Divisions of Allergy and Immunology, the Department of Medicine, Children’s Hospital Medical Center and the Department of Pediatrics, Harvard Medical School. This work was supported by grants from the Charles H. Hood Foundation and United States Public Health Service, Grant AI 05877, and by the Dooner Company and the National Foundation March of Dimes. Received for publication July 11, 1978. Accepted for publication April 10, 1979. Reprint requests to: Dr. Raif S. Geha, Allergy Division, Children’s Hospital Medical Center, 300 Longwood Ave., Boston, MA 02115. *Recipient of Medical Foundation Award No. 1553. Vol. 64, No. 3, pp. 182-188
terenol are mediated via 3’, 5’-cyclic adenosine monophosphate* (CAMP), and several investigators3-7, ‘* have demonstrated reduced CAMP production in leukocytes from asthmatic subjects following exposure to beta adrenergic agents. Agents that modulate intracellular CAMP have been shown to affect the ability of lymphocytes to form rosettes with sheep red cells (E) and the sheep red cell intermediate EAC 1423 (EAC3). Chisari and Edington13 and Galant and Remo14 have observed inhibition of lymphocyte-sheep red blood cell rosette formation (E rosette) following exposure of human lymphocytes to agents that increase intracellular CAMP. Lang et a1.8recently demonstrated that blunted inhibition of E rosette formation by lymphocytes from asthmatics followed incubation with beta agonists. Ito et al. l5 demonstrated enhancement of EAC3 rosette formation following exposure of mouse spleen cells to beta adrenergic
VOLUME NUMBER
TABLE
Pharmacologic effects on EAC3 rosettes
64 3
I. Effect of isuprel
and theophylline
183
on EAC3 rosette formation % EAC3 rosettes*
Agent
added
Adult normal (n = 30)
Age- and sex-matched controls (n = 11)
13.2 ? 4.0
None
14.6 ? 4.9
15.1 +- 3.0
Isoproterenol, lO-9 M
20.1 ? 5.5 A + 6.9 (
21.9 ? 3.5 A + 6.8 (
lOmyM
20.2 L 5.8 A + 7.0 (
21.1 -+ 3.6 A6.0 (CO.01)
Theophylline,
Asthma (n = 35)
Allergic rhinitis (n = 10)
14.4 -t 3.2
14.6 + 4.9 A = 0 (>O.l)
21.6 f 3.3 A + 7.2 (
14.3 + 4.5 A - 0.3 (~0.01)
21.9 i- 3.2 A + 7.5 (
*Mean percent EAC3-RFC 2 SD.
t Mean change from baseline, A; p values shown in parentheses. agents. The present article describes the modulation of EAC3 rosette formation in normal and asthmatic subjects by agents that increase intracellular CAMP.
MATERIAL Subjects
AND METHODS
Thirty-live patients with asthma, aged 3 to 19 yr, were selected from the Allergy Clinic at the Children’s Hospital Medical Center. The objectives of the study were explained to the patients and their parents and consent was obtained. Of the 35 asthmatics, 30 had allergic or mixed asthma documented by history, positive immediate skin tests to at least two allergens, and elevated levels of serum immunoglobulin E (IgE); and five had intrinsic asthma documented by history, absence of positive immediate skin tests, and low levels of serum IgE. The ages of the patients ranged from 5 to 21 yr. All were symptom free at the time of the study. Ten asthmatics had received no therapy for 2 wk or more. Of these, three had been free of bronchospasm and bronchodilator therapy had not been necessary for a period of 5 yr. Fifteen of the patients with asthma had received only theophylline (20 to 26 mg/kg/24 hr) therapy during the month prior to the study. The remaining IO asthmatics were all receiving daily theophylline and metaptoterenol (20 to 60 mgi24 hr) and six of them were also receiving altemateday prednisone. Control groups consisted of (1) 30 healthy adults selected from laboratory and hospital personnel, with an age range of 19 to 3 l yr; (2) 11 healthy children, aged 4 to 16 yr and sex matched with the asthma group; and (3) 10 patients with allergic rhinitis, aged 8 to 17 yr, who had positive immediate skin tests, elevated levels of serum IgE, and normal pulmonary function tests, and who had not received any medications for at least 2 wk prior to the study. None of the control subjects had received any medication within 2 wk of the study. Three adult control subjects were studied before and after administration of theophylline, 1 gm daily for 2 days.
Reagents Ficoll-Hypaque, Sephadex G-200, and Sepharose 4B were obtained from Pharmacia, Piscataway, N. J. RPMI1640, fetal calf serum (FCS), and sheep red blood cells (E)
were purchased from Grand Island Biological Co., Grand Island, N. Y. Rabbit anti-sheep red cell hemolysin and rabbit anti-human Fab were from Microbiological Associates, Baltimore, Md. Isoproterenol hydrochloride (I), theophylline (Th), propranolol hydrochloride (P), adrenaline hydrochloride (A), puromycin hydrochloride (PU), noradrenalin (NA), HEPES, calcium edetate (EDTA), and ammonium chloride were obtained from Sigma Chemical Co., St. Louis, MO. Fluorescein-conjugated goat antisera specific to the heavy chains of human IgD and IgM were purchased from Meloy Chemicals, Springfield, Va. Methylprednisolone (Solu-medrol) was a product of Upjohn Chemical Co., Kalamazoo, Mich. Cholera toxin (CT) was kindly supplied by Dr. Peter Eccheveria, Division of Infectious Diseases, Children’s Hospital Medical Center, Boston, Mass. Prostaglandins El, EY, and Fza (PGE,, PGEZ, PGF& were kindly provided by Dr. John Pike, Upjohn Chemical Co. All reagents were freshly prepared the day of the study. EAC3 reagent was prepared by first incubating sheep red cells with a 1: 1,000 dilution of a rabbit anti-sheep red cell stroma IgM antibody (Behring Werke, Somerville, N. J.) for 1 hr at room temperature, washing the red cells, and then incubating them at 37” C with a I : 40 dilution of freshfrozen normal human semm preabsorbed with sheep red cells. The final cell suspension was washed in Hanks’ balanced salt solution (HBSS) and used within 3 days.
Isolation
of peripheral
blood lymphocytes
Mononuclear cells were separated from heparinized blood over gradients of Ficoll-Hypaque,” washed three times in HBSS, and suspended in RPMI-1640 culture medium containing 15% FCS. The cells were incubated overnight in plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.) to allow adherent cells to stick to the surface of the flask. The next day the nonadherent cells were collected and washed three times in HBSS prior to use.
EAC3 rosette Washed lymphocytes resuspended in HBSS-FCS, at a concentration of 8 X IO6 cells/O. 1 ml, were incubated with an equal volume of a 1% suspension of the EAC3 at 37” C for 15 min. Cells were spun at 200 X g for 10 min and then
184
J. ALLERGY
Kohn et al.
ADULT NORMAL
ALLERGIC RHINITIS ASTHMA
i “a
i7 B-BLANK I - ISOPROTERNOL FIG. 1. Effect of EAC3 rosette formation by lymphocytes from normal subjects (adult and children) and from patients with allergic rhinitis and asthma. Lymphocyte suspensions were incubated with I for 1 hr prior to the EAC3 rosette assay. Percent EAC3 rosette formation is shown on the ordinate. 8, Baseline (incubated with phosphatebuffered saline); I, isoproterenol (lOeg M).
left at room temperature for 10 min. The pellets were then resuspended vigorously and an aliquot of each cell suspension was placed in an improved Neubauer hemacytometer. A minimum of 100 cells were counted and the percent rosetted cells was recorded. Rosette-forming cells were defined as lymphocytes with three or more adherent erythrocytes.
lmmunofluorescence Eight million cells were suspended in 50 ~1 cold HBSS containing 5% fetal calf serum and 0.1% sodium azide, gently mixed with 50 ~1 fluorescein-conjugated goat antisera, and incubated at 4” C for I hr. Antisera to IgM and IgD were used since IgM and IgD are the major surface immunoglobulins of human B-lymphocytes.‘7 The cell suspension was layered over 1 ml FCS and spun in the cold (4” C) state at 200 x g for 10 min. Then the cells were washed twice in cold, azide-containing HBSS medium, resuspended in 100 ~1 medium, and placed on a glass slide. The percent of cells exhibiting membrane fluorescence was assessed using a Zeiss Orthoplan fluorescent microscope.
Effect of drugs on EAC3 rosette
formation
I, Th, CT, PGE,, PGE2, PGF,,, A, and NA. Fresh serial lOO-fold dilutions of these agents in phosphate-buffered saline were prepared for each experiment and incubated with lymphocytes as described. Methylprednisolone or propranolol. Lymphocytes were preincubated for 15 min with methylprednisolone (10m3 M
CLIN. IMMUNOL. SEPTEMBER 1979
to 10e7 M) or propranolol (lo-” M to 10 -I’ M) prior to their incubation with I, Th, or A. Puromycin. PU was added to the cell suspensions 15 min prior to adding I or Th and allowed to remain in the culture medium until all drugs were washed away just prior to EAC3 rosetting.
lmmunosorbent
column
preparation
Rabbit anti-human Fab was purified over a Sepharose 4B immunoabsorbent column containing human gamma globulin purified from normal serum by diethylaminoethyl cellulose chromatography. The antibody was eluted with 3 M sodium thiocyanate and dialyzed extensively against 0.1 M sodium bicarbonate prior to cross-linking to Sephadex G-200 beads. Sephadex G-200 was sieved to achieve uniform bead size (88 to 120 p), swollen in water, and activated with cyanogen bromide.” The activated Sephadex was gently mixed with purified rabbit antibody for 4 hr and then exhaustively washed. Approximately 0.25 to 0.35 mg protein was bound per milliliter of activated Sephadex. Disposable lo-ml syringes were fitted with polyethylene disks (Bell-Art Products, Panawock, N. J.) and packed with 5 ml Sephadex conjugate. IR The column was washed with HBSS-FCS prior to use.
Cell separation
studies
Lymphocytes were resuspended at a concentration of 20 x lo6 cells/ml in cold RPMI-1640 containing 10% FCS, calcium EDTA (0.092%), and 0.12 M HEPES and drops were applied to the 5-ml Sephadex anti-human Fab column. Nonretained cells were collected by stepwise elution with 15-ml aliquots of starting medium at a flow rate of 0.5 ml/min until the effluent was virtually cell free. Bound cells were recovered by gentle agitation and elution with two 15ml aliquots of medium containing 10 mg/ml human gamma globulin. The resulting cell suspension was enriched in B cells; 67% were EAC3 rosette-forming cells (EAC3RFC) while only 6% were E rosette- forming cells (E-RFC). The nonretained population was further fractionated into E rosette-positive and E rosette-negative cells. in brief, 10 ml lymphocyte suspension (4 x 10” cells/ml HBSS) were incubated with 1 ml 5% E and 1.5 ml FCS for 10 min at 37” C in an atmosphere of 5% COB in air. Cells were pelleted at 200 x g for 5 min in a 4” C centrifuge and incubated at 4” C for 1 hr. The pellet was gently resuspended, layered over a Ficoll-Hypaque gradient, and centrifuged at 400 x g for 30 min at room temperature. Cells that floated at the top of the gradient were designated null cells and contained less than 5% E-RFC and 20% to 30% EAC3-RFC. Cells that sedimented to the bottom of the gradient together with the sheep red cells were resuspended in 5 ml 0.87% ammonium chloride solution for 5 min at 37” C to lyse the red cells, and then were immediately washed with HBSS-FCS. The resulting lymphocyte suspension was designated T cells and contained more than 90% E-RFC and less than 6% EAC3-RFC. Unfractionated cells, T, B, and null cells were washed three times with HBSS-FCS prior to rosetting with EAC3.
VOLUME NUMBER
Pharmacologic
64 3
RESULTS Effect of I and Th on EAC3 rosette
effects
on EAC3 rosettes
185
formation
Baseline EAC3 rosette formation was similar for all groups (Table I). Preincubation of normal peripheral blood lymphocytes (PBL) with I and Th for 1 hr resulted in a statistically significant increase in the percent EAC-RFC (Table I and Fig. 1). Mean increase of EAC3-RFC averaged 49% with I and 47% with Th for the two control groups of healthy subjects. Identical results were obtained when the incubation time of lymphocytes with these drugs was varied from a minimum 15 min to a maximum 3 hr. Maximal increase in EAC3-RFC occurred at a concentratitn of lo-” M I, IO-” M Th, and 1 rig/ml CT (Fig. 2, u-c). In contrast, EAC3 rosette formation by lymphocytes from patients with asthma was not significantly affected by preincubation with I, Th, or CT in doses covering the range of lop3 M to lo-l5 M (data not shown). Response of lymphocytes from 10 patients with allergic rhinitis was similar to that of normal lymphocytes, with mean increases in EAC3-RFC of 50% and 52%, following incubation with I and Th, respectively. The observed increase in the percent of normal EAC-RFC following incubation with I and Th reflects a true increase in the number of complement receptor-bearing lymphocytes, since cell counts and cell viability (by trypan blue exclusion) were identical for control and drug-treated cell suspensions of both normal and asthmatic subjects. Increases in EAC3 rosette formation by PBL were unaffected by treatment of normal donors with Th (1 gm daily for 2 days) or by overnight incubation of normal cells in serum (at a final concentration of 10% in RPMI-1640 medium) obtained from patients with asthma. Preincubation of lymphocytes from asthmatic subjects with methylprednisolone ( 10m3M to 10e7 M for 15 min) did not reverse the resistance of these lymphocytes to the enhancing effect of I and Th on EAC3 rosette formation (data not shown). Effect of fetal calf serum FCS from Grand Island Biological Co., Lots A475416 and A272325, was used throughout the present study. In preliminary studies it was found that in the presence of two other lots of FCS, G.I.B.CO. A775524 and A979117, EAC3 rosette formation by normal lymphocytes was not enhanced by I (mean change in nine experiments, + 1.1% whereas EAC3 rosette formation by lymphocytes from patients with asthma was decreased (mean change in nine experiments, -4.8%. In order to best study the effects of various agents that increase intracellular CAMP on
5?----3 5 7
9
II 13 I5
-
CONCENTRATION
-0 lOXMOLAR
FIG. 2. Dose-response effect of agents that tracellular CAMP levels on EAC3 rosette normal lymphocytes. Percent EAC3 rosette shown on the ordinate. Drug concentration the abscissa. Results represent mean k SD imentsa, Isoproterenol; b, theophylline; c, d, PGE,; e, adrenaline; t noradrenalin.
modulate information by formation is is shown on of five expercholera toxin;
EAC3 rosette formation in normal lymphocytes, FCS from lots supporting the I-mediated increase in normal EAC-RFC was used. Dialysis and heat treatment (60” C for 1 hr) of FCS did not affect its permissive effect on I-mediated enhancement of EAC3 rosette formation. Response of normal lymphocytes that modulate cAMP levels
to drugs
I, Th, CT, PGEl, and A all caused increases in EAC3 rosette formation by normal lymphocytes (Fig. 2, u-e). Propranolol, PGEz, and PGFz, did not affect EAC3 rosette formation (data not shown). Noradrenalin produced inhibition of EAC3 rosette formation (Fig. 2,f). This inhibition was not augmented by the addition of propranolol. Propranolol partially blocked the enhancing effects of I and A but not those of Th (Fig. 3, a-c). Effect of puromycin on l-mediated enhancement of EAC3 rosette formation Preincubation of lymphocytes with PU blocked I-mediated enhancement of EAC3 rosette formation. This effect was partial at a PU concentration of 1 pg/ml and complete at 10 ,ug/ml (Fig. 4). Response
of E-WC to I
Preincubation with I did not cause a change in the percent of E-RFC of lymphocytes from eight normal (before, 53.9 t 8.9; after, 54.7 * 8.8) and eight asthmatic (before, 57.6 ? 6.9; after, 56.7 + 6.8) subjects.
186
J. ALLERGY
Kohn et al.
CLIN. IMMUNOL. SEPTEMBER 1979
70s
f
-9 b, ADRENALINE lo-% MI OL---L---L--0 35791113
0
CONCENTRATION
357911
c 1 THEOPHYLLINE l&4
0
357911
OF PROPANOLOL IO-’ MOLAR
FIG. 3. Effect of propranolol on the enhancement of EAC3 rosette formation by (al IO-” M isoproterenol; lb) 1O-9 M adrenaline, and (cl 10m9 M theophylline. Percent EAC3 rosette formation is shown in the ordinates. Concentrations of propranolol are shown on the abscissa. The * represents the baseline value of EACB-RFC in the absence of all drugs.
W
‘;----I.,,, F
to-
a
5
El
T
IB 9
CONTROLS
I HULL I
-B I B I “NFRACTIONATED NULL I t ASTHMATIC
FIG. 5. Enhancement of EAC3 rosette formation by I in lymphocyte subpopulations of normal and asthmatic subjects. Lymphocyte subpopulations were incubated with I (10m9 M) for 1 hr prior to the EAC3 rosette assay. Percent EAC3 rosette formation is shown on the ordinate.
DISCUSSION
15.
l5 o
IB
following incubation of either unfractionated or null cells from control subjects.
w” 25. L ii 20B s a
-~-B IB LIHPnOCITE “NFRACPOPULPlTlONTIONATED I
---++
M X----X e----e
CONCENTRATION
ISOPROTERENOL ISOPROTERENOL+PUROMYCIN ( Irq/ml) ISOPROTERENOL+ PUROMYCIN (Ioyg/ml) OF ISOPROTERENOL
16” MOLAR
FIG. 4. Effect of puromycin on l-mediated enhancement of EAC3 rosette formation by normal lymphocytes. Percent EAC3 rosette formation is shown on the ordinate. Concentration of I is shown on the abscissa. Results represent mean -C SD of four experiments. The A represents the baseline percent EAC-RFC in the absence of all drugs.
Enhancement of EAC3 rosette I in lymphocyte subpopulations
formation
by
T cell-rich and B cell-rich subpopulations from normal individuals failed to demonstrate a significant increase in EAC3-RFC following preincubation with I; on the other hand the null cell population demonstrated a 50% increase (Fig. 5). Neither unfractionated nor null cell populations from asthmatic patients responded to I. Effect of I and Th on expression membrane immunoglobulins
of
No increase in the number of surface immunofluorescent staining cells was seen with I or Th
Expression of a surface receptor for the third component of complement (C3) is a characteristic of most of the B-lymphocytes as well as of 20% to 30% of the null lymphocytes. lg Modulation of C3 receptor expression by adrenergic agents has recently been reported by Ito et al. l5 who demonstrated that beta adrenergic agents increased EAC3 rosette formation in mouse spleen cells. The present article describes the enhancement of EAC3 rosette formation in normal human PBL by agents that increase intracellular CAMP, and demonstrates the lack of this enhancement in lymphocytes from subjects with asthma. Early in our studies of normal lymphocytes it became clear that only certain lots of FCS permitted I-mediated enhancement of EAC3 rosette formation. In the presence of other lots of FCS, EAC3 rosette formation was not affected by the addition of I. These results suggested the presence of a serum factor in certain lots of FCS which exerts a permissive effect on I-mediated increases in EAC3 rosette formation. This factor was demonstrated to be heat stable and nondialyzable (> 10,000 mw) and thus it is unlikely to be a prostaglandin. Similar variability in the capacity of different lots of FCS to support in vitro immune responses has been well documented.” To best examine the EAC3 response of normal lymphocytes to various agents that increase intracellular CAMP, we used only those lots of FCS which supported the l-mediated enhancement of normal lymphocyte EAC3 rosette formation.
VOLUME NUMBER
64 3
The beta adrenergic agent isoproterenol significantly increased EAC3-RFC in normal lymphocytes (Fig. 1, Table I) with an optimum concentration of lo-” M (Fig. 2, a). Lymphocytes from asthmatic subjects failed to show an increase after incubation with a broad range (lo-” M to 10-l” M) of I concentrations. Similar findings were observed with Th (Fig. 2, b, and Table I), CT, PGEl, and A (Fig. 2). The beta adrenergic antagonist propranolol blocked the enhancement of EAC-RFC induced by I and A (Fig. 3, a and h) but did not affect the enhancement of EAC3RFC induced by Th (Fig. 3, L.). All pharmacologic agents that caused enhancement of EAC3 rosette formation are known to cause an increase in intracellular CAMP.“” ” Intracellular CAMP levels were not measured. However, the present data suggest that the observed enhancement of EAC3 rosette formation is mediated through an increase in intracellular CAMP and/or through CAMP-triggered events and not solely through selective triggering of beta adrenergic receptors. The observed enhancement of EAC3 rosette formation in normal lymphocytes reflects an increase in the actual number of lymphocytes expressing complement receptors, since cell counts and viability were identical in control and in drug-incubated cell suspensions and since macrophages had been removed by overnight incubation of cells in plastic flasks. Furthermore, the data suggests that the increase in complement receptor- bearing lymphocytes is an active expression of new receptors and not merely an uncovering of already present receptors. Puromycin did not alter baseline EAC3 rosette formation but blocked I-mediated enhancement (Fig. 4) of EAC3-RFC, suggesting that de novo protein synthesis is required for the expression of new lymphocyte complement receptors. Cells expressing new EAC3 receptors were demonstrated to reside in the null cell fraction (Fig. 5). Such cells could not be detected in the null cell fraction from asthmatic patients. Expression of new EAC3-EFC was complete within 15 mitt, was stable for more than 3 hr, and was not accompanied by an increase in surface immunoglobulin-bearing cells . Our data demonstrate a fundamental difference between the response of normal lymphocytes and lymphocytes from asthmatics to I in the EAC3 rosetteforming assay regardless of the source of FCS. When permissive lots of FCS were used in the assay, I enhanced EAC3 rosette formation by lymphocytes from normal subjects and did not affect EAC3 rosette formation by lymphocytes from asthmatic patients over the entire dose range used ( 10e3M to lo-l5 M). When
Pharmacologic
effects
on EAC3 rosettes
187
nonpermissive lots of FCS were used, addition of I did not affect EAC3 rosette formation by normal lymphocytes and decreased the percent of EAC-RFC in lymphocytes from asthmatics. Several investigators 2, 5, 6. IO. 23-26 have o,,served that a variety of cells from patients with asthma demonstrate an altered sensitivity to beta adrenergic agents. This altered sensitivity of lymphocytes from asthmatics has been ascribed to beta adrenergic blockade, a theory first proposed by Szentivanyi.’ Thus Parker et al.“, 6, 26 and Logsdon et al.” have reported that leukocytes and lymphocytes from patients with asthma produce less CAMP than normal leukocytes and lymphocytes following incubation with catecholamines, and further demonstrated that the altered response of these cells can be reversed by in vivo3, 26or in vitro26 corticosteroid administration. On the other hand, other investigators have observed a diminished CAMP response of lymphocytes from asthmatics only during active bronchospasm.4. 9. l2 Conolly and Greenacre” and Morris et al.’ presented evidence that this diminished response was due to ongoing therapy with beta adrenergic agents and was not reversed by corticosteroids administered in viva.’ To control for all these factors, asthmatic patients included in the present study had normal lung function at the time of testing. Twenty-five of the 35 asthmatics had not received any beta adrenergic agents within 6 wk of the study, including seven patients who were receiving no medications at all and three patients who had had no bronchospasm or bronchodilator therapy for more than 5 yr. The remaining 10 asthmatic patients were receiving daily theophylline and metaproterenol and six of them were also receiving alternate-day corticosteroid therapy. In no case did medications affect the resistance of lymphocytes from patients with asthma to the enhancing effect of I, Th, and CT on EAC3 rosette formation; and lymphocytes from asthmatics continued to demonstrate resistance to the enhancement of EAC3 rosette formation following in vitro preincubation with methylprednisolone. Furthermore, I- and Th-mediated enhancement of EAC3 rosette formation in normal lymphocytes was unaffected by pretreatment of normal donors with theophylline. Lymphocytes from asthmatic patients also failed to respond to agents that increase intracellular CAMP by Bechanisms other than stimulation of the lymphocyte beta receptor. Thus, while normal lymphocytes responded to Th, CT, and PGEl with an increase in EAC3 rosette formation of a magnitude similar to that caused by I, lymphocytes from asthmatic subjects were totally resistant to the EAC3-enhancing effect of
188
Kohn et al.
J. ALLERGY
these agents. Furthermore, it was demonstrated that this resistance was intrinsic to these lymphocytes since incubation of normal lymphocytes with sera from asthmatic subjects did not affect the response of the normal cells. Since intracellular CAMP was not measured, the present findings suggest but do not conclusively prove that the CAMP response of the lymphocytes to the pharmacologic agents is abnormal in asthma. The differential response of lymphocytes from normal and asthmatic subjects to I and Th can be used as an adjunct in the investigation of patients with reversible airway disease. Studies of infants who are at risk of developing asthma (patients with bronchiolitis, offspring of asthmatic parents) are currently underway to determine if the lymphocyte abnormality described in the present report precedes the development of asthma. The authors are grateful to Dr. Fred Rosenfor reviewing the manuscript, Mr. Scott Ames and Ms. Lilya Naiman for technical assistance,and Miss Cathy Clark for secretarial assistance. REFERENCES 1. Szentivanyi A: The beta-adrenergic theory of atopic abnormality in bronchial asthma. J ALLERGY CLIN IMMUNOL 42~203, 1968. 2. Smith JW, Steiner AL, Newberry WM, Parker CW: Cyclic adenosine 3’) 5’-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation. J Clin Invest 50~432, 1971. 3. Logsdon PJ, Middleton E Jr, Coffey RG: Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and nonasthmatic subjects. J ALLERGY CLIN IMMUNOL 50~45,
1972.
4. Alston WC, Pate1 RK, Kerr JW: Response of leucocyte adenyl cyclase to isoprenaline and effect of alpha-blocking drugs in extrinsic bronchial asthma. Br Med J 1:90, 1974. 5. Parker CW, Smith JW: Alteration in cyclic adenosine monophosphate metabolism in human bronchial asthma. J Clin lnvest 52:48, 1973. 6. Parker CW, Baumann ML, Huber MC? Alterations in cyclic AMP metabolism in human bronchial asthma. J Clin Invest 52: 1336, 1973. 7. Morris HG, Rusnak SA, Selner JC, Barzens K, Barnes J: Comparative effect of ephedrine on adrenergic responsiveness in normal and asthmatic subjects. J ALLERGY CLIN IMMUNOL 61:294, 1978. 8. Lang P, Goel Z, Grieco MH: Subsensitivity of T lymphocytes to sympathomimetic and cholinergic stimulation in bronchial asthma. J ALLERGY CLIN IMMUNOL 61:248, 1978.
CLIN. IMMUNOL SEPTEMBER 1979
9. Gillespie E, Valentino MD, Lichtenstein LM: Cyclic AMP metabolism in asthma: Studies with leukocytes and lymphocytes J ALLERGY CLIN IMMUNOL 53~27, 1974. 10. Logsdon PJ, Carnright DV, Middleton E Jr, Coffey RG: The effect of phentolamine on adenylate cyclase and on isoprotereno1 stimulation in leukocytes from asthmatic and nonasthmatic subjects. J ALLERGY CLIN IMMUNOL 52: 148. 1973. Il. Conolly ME, Greenacre JK: The lymphocyte beta-adrenoceptor in normal subjects and patients with bronchial asthma. J Clin Invest 58: 1307, 1976. 12. Pate1 KR, Alston WC, Kerr JW: The relationship of leucocyte adenyl cyclase activity and airway response to beta blockade and allergen challenge in extrinsic asthma. Clin Allergy 4:3 I I. 1974. 13. Chisari FV, Edington TS: Human T lymphocyte “E” rosette function. J Exp Med 140: 1122, 1974. 14. Galant SP, Remo RA: Beta adrenergic inhibition of human T lymphocyte rosettes. J lmmunol 114:512, 197.5. 15. Ito M, Sless F, Parrott DMV: Evidence for control of complement receptor rosette forming cells by alpha and betaadrenergic agents. Nature 266:633, 1977. 16 Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Lab Invest 21 (suppl. 97):77. 1968. 17 Fu SM, Winchester RJ, Kunkel HG: Occurrence of surface IgM, IgD and free light chains on human lymphocytes. J Exp Med 139451, 1974. 18. Chess L, Mac Dermott RP, Schlossman SF: Immunologic functions of isolated human lymphocyte subpopulations. J lmmunol 113: 1113, 1974. 19. Chess L. Schlossmann SF: Human lymphocyte subpopulations. Adv lmmunol 25:213, 1977. 20. Dalton RW, Eady JD: An in vitro system for the study of the mechanism of antigenic stimulation in the secondary response. Immunology 7:40, 1964. 21. Bourne HR, Melmon KL: Adenyl cyclase in human leukocytes: evidence for activation by separate beta adrenergic and prostaglandin receptors. J Pharmacol Exp Ther 178: I, 197 1. 22. Lichtenstein LM, Henney CS. Bourne HR, Greenough WB Ill: Effects of cholera toxin on in vitro models of immediate and delayed hypersensitivity. J Clin Invest 52691, 1973. 23. Grieco MH, Pierson RN Jr, Pi-Srenyer FX: Comparison of the circulatory and metabolic effects of isoproterenol. epinephrine and methoxamine in normal and asthmatic subjects. Am J Med 4.4~863, 1963. 24. Lackey SD Jr, Glennon JA, Reed CE: Comparison of some metabolic responses in normal and asthmatic subjects to epinephrine and glucagon. J Allergy 40: 349, 1967. 25. Schwartz HT, White LW: Urinary and plasma cyclic AMP responses to epinephrine in asthmatic and normal subjects. J ALLERGY
CLIN IMMUNOL
51:88,
1973.
26. Parker CW, Huber MG, Baumann ML: Alterations in cyclic AMP
metabolism
52: 1342, 1973.
in human bronchial
asthma.
J Clin
Invest
We studied the effect of isoproterenol hydrochloride (I) and theophylline (Th) on EAC3 rosette formation by lymphocytes from 30 normal adult subjects, 35 asthmatic subjects, 11 subjects who were age and sex matched with the asthma group, and 10 subjects with allergic rhinitis. Baseline EAC3 rosette numbers were similar for all groups (normal adults, 13.2%; normal children, 15.190; rhinitis, 14.490; asthma, 14.6%). Incubation with I and Th in the presence of fetal calf serum caused enchancement of EAC3 rosette formation by lymphocytes from normal adults (mean increase, 52% with I and 53% with Th), from normal age- and sex-matched controls (mean increase, 45% with I and 4090 with Th), f rom subjects with allergic rhinitis (mean increase, 50% with I and 52% with Th), but not by lymphocytes from asthmatic subjects (mean increase, 0% with I and 290 with Th). These results were not affected by bronchodilator therapy. Similar results were obtained with cholera toxin, prostaglandin E,, and adrenaline, suggesting that the defect in lymphocytes from asthmatic patients resides at the level of 3’, 5’qclic adenosine monophosphate (CAMP) production or at the level of CAMP-triggered events. Lymphocyte fractionation experiments into T cell-rich, B cell-rich, and null cell populations revealed that the null cell population was a target for the l-mediated enhancement of EAC3 rosette formation. Null cells from normal subjects, but not from asthmatic subjects, exhibited an increase in the number of EAC3 rosettes formed following incubation with I. This enhancement required protein synthesis as it was prevented by the addition of puromycin hydrochloride. The resistance of peripheral blood lymphocytes from asthmatics to the enhancing effect of I and Th on EAC3 rosette formation may be used to study the biochemical defect in asthma and should be assessed for possible use in the detection of the latent asthmatic.
Beta adrenergic blockade was first postulated by Szentivanyi 1 to explain the defect in bronchial asthma. Subsequent studies have suggested that asthmatics have altered metabolic and cellular responses to isoproterenol. *-l* The adrenergic effects of isopro-
From the Divisions of Allergy and Immunology, the Department of Medicine, Children’s Hospital Medical Center and the Department of Pediatrics, Harvard Medical School. This work was supported by grants from the Charles H. Hood Foundation and United States Public Health Service, Grant AI 05877, and by the Dooner Company and the National Foundation March of Dimes. Received for publication July 11, 1978. Accepted for publication April 10, 1979. Reprint requests to: Dr. Raif S. Geha, Allergy Division, Children’s Hospital Medical Center, 300 Longwood Ave., Boston, MA 02115. *Recipient of Medical Foundation Award No. 1553. Vol. 64, No. 3, pp. 182-188
terenol are mediated via 3’, 5’-cyclic adenosine monophosphate* (CAMP), and several investigators3-7, ‘* have demonstrated reduced CAMP production in leukocytes from asthmatic subjects following exposure to beta adrenergic agents. Agents that modulate intracellular CAMP have been shown to affect the ability of lymphocytes to form rosettes with sheep red cells (E) and the sheep red cell intermediate EAC 1423 (EAC3). Chisari and Edington13 and Galant and Remo14 have observed inhibition of lymphocyte-sheep red blood cell rosette formation (E rosette) following exposure of human lymphocytes to agents that increase intracellular CAMP. Lang et a1.8recently demonstrated that blunted inhibition of E rosette formation by lymphocytes from asthmatics followed incubation with beta agonists. Ito et al. l5 demonstrated enhancement of EAC3 rosette formation following exposure of mouse spleen cells to beta adrenergic
VOLUME NUMBER
TABLE
Pharmacologic effects on EAC3 rosettes
64 3
I. Effect of isuprel
and theophylline
183
on EAC3 rosette formation % EAC3 rosettes*
Agent
added
Adult normal (n = 30)
Age- and sex-matched controls (n = 11)
13.2 ? 4.0
None
14.6 ? 4.9
15.1 +- 3.0
Isoproterenol, lO-9 M
20.1 ? 5.5 A + 6.9 (
21.9 ? 3.5 A + 6.8 (
lOmyM
20.2 L 5.8 A + 7.0 (
21.1 -+ 3.6 A6.0 (CO.01)
Theophylline,
Asthma (n = 35)
Allergic rhinitis (n = 10)
14.4 -t 3.2
14.6 + 4.9 A = 0 (>O.l)
21.6 f 3.3 A + 7.2 (
14.3 + 4.5 A - 0.3 (~0.01)
21.9 i- 3.2 A + 7.5 (
*Mean percent EAC3-RFC 2 SD.
t Mean change from baseline, A; p values shown in parentheses. agents. The present article describes the modulation of EAC3 rosette formation in normal and asthmatic subjects by agents that increase intracellular CAMP.
MATERIAL Subjects
AND METHODS
Thirty-live patients with asthma, aged 3 to 19 yr, were selected from the Allergy Clinic at the Children’s Hospital Medical Center. The objectives of the study were explained to the patients and their parents and consent was obtained. Of the 35 asthmatics, 30 had allergic or mixed asthma documented by history, positive immediate skin tests to at least two allergens, and elevated levels of serum immunoglobulin E (IgE); and five had intrinsic asthma documented by history, absence of positive immediate skin tests, and low levels of serum IgE. The ages of the patients ranged from 5 to 21 yr. All were symptom free at the time of the study. Ten asthmatics had received no therapy for 2 wk or more. Of these, three had been free of bronchospasm and bronchodilator therapy had not been necessary for a period of 5 yr. Fifteen of the patients with asthma had received only theophylline (20 to 26 mg/kg/24 hr) therapy during the month prior to the study. The remaining IO asthmatics were all receiving daily theophylline and metaptoterenol (20 to 60 mgi24 hr) and six of them were also receiving altemateday prednisone. Control groups consisted of (1) 30 healthy adults selected from laboratory and hospital personnel, with an age range of 19 to 3 l yr; (2) 11 healthy children, aged 4 to 16 yr and sex matched with the asthma group; and (3) 10 patients with allergic rhinitis, aged 8 to 17 yr, who had positive immediate skin tests, elevated levels of serum IgE, and normal pulmonary function tests, and who had not received any medications for at least 2 wk prior to the study. None of the control subjects had received any medication within 2 wk of the study. Three adult control subjects were studied before and after administration of theophylline, 1 gm daily for 2 days.
Reagents Ficoll-Hypaque, Sephadex G-200, and Sepharose 4B were obtained from Pharmacia, Piscataway, N. J. RPMI1640, fetal calf serum (FCS), and sheep red blood cells (E)
were purchased from Grand Island Biological Co., Grand Island, N. Y. Rabbit anti-sheep red cell hemolysin and rabbit anti-human Fab were from Microbiological Associates, Baltimore, Md. Isoproterenol hydrochloride (I), theophylline (Th), propranolol hydrochloride (P), adrenaline hydrochloride (A), puromycin hydrochloride (PU), noradrenalin (NA), HEPES, calcium edetate (EDTA), and ammonium chloride were obtained from Sigma Chemical Co., St. Louis, MO. Fluorescein-conjugated goat antisera specific to the heavy chains of human IgD and IgM were purchased from Meloy Chemicals, Springfield, Va. Methylprednisolone (Solu-medrol) was a product of Upjohn Chemical Co., Kalamazoo, Mich. Cholera toxin (CT) was kindly supplied by Dr. Peter Eccheveria, Division of Infectious Diseases, Children’s Hospital Medical Center, Boston, Mass. Prostaglandins El, EY, and Fza (PGE,, PGEZ, PGF& were kindly provided by Dr. John Pike, Upjohn Chemical Co. All reagents were freshly prepared the day of the study. EAC3 reagent was prepared by first incubating sheep red cells with a 1: 1,000 dilution of a rabbit anti-sheep red cell stroma IgM antibody (Behring Werke, Somerville, N. J.) for 1 hr at room temperature, washing the red cells, and then incubating them at 37” C with a I : 40 dilution of freshfrozen normal human semm preabsorbed with sheep red cells. The final cell suspension was washed in Hanks’ balanced salt solution (HBSS) and used within 3 days.
Isolation
of peripheral
blood lymphocytes
Mononuclear cells were separated from heparinized blood over gradients of Ficoll-Hypaque,” washed three times in HBSS, and suspended in RPMI-1640 culture medium containing 15% FCS. The cells were incubated overnight in plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.) to allow adherent cells to stick to the surface of the flask. The next day the nonadherent cells were collected and washed three times in HBSS prior to use.
EAC3 rosette Washed lymphocytes resuspended in HBSS-FCS, at a concentration of 8 X IO6 cells/O. 1 ml, were incubated with an equal volume of a 1% suspension of the EAC3 at 37” C for 15 min. Cells were spun at 200 X g for 10 min and then
184
J. ALLERGY
Kohn et al.
ADULT NORMAL
ALLERGIC RHINITIS ASTHMA
i “a
i7 B-BLANK I - ISOPROTERNOL FIG. 1. Effect of EAC3 rosette formation by lymphocytes from normal subjects (adult and children) and from patients with allergic rhinitis and asthma. Lymphocyte suspensions were incubated with I for 1 hr prior to the EAC3 rosette assay. Percent EAC3 rosette formation is shown on the ordinate. 8, Baseline (incubated with phosphatebuffered saline); I, isoproterenol (lOeg M).
left at room temperature for 10 min. The pellets were then resuspended vigorously and an aliquot of each cell suspension was placed in an improved Neubauer hemacytometer. A minimum of 100 cells were counted and the percent rosetted cells was recorded. Rosette-forming cells were defined as lymphocytes with three or more adherent erythrocytes.
lmmunofluorescence Eight million cells were suspended in 50 ~1 cold HBSS containing 5% fetal calf serum and 0.1% sodium azide, gently mixed with 50 ~1 fluorescein-conjugated goat antisera, and incubated at 4” C for I hr. Antisera to IgM and IgD were used since IgM and IgD are the major surface immunoglobulins of human B-lymphocytes.‘7 The cell suspension was layered over 1 ml FCS and spun in the cold (4” C) state at 200 x g for 10 min. Then the cells were washed twice in cold, azide-containing HBSS medium, resuspended in 100 ~1 medium, and placed on a glass slide. The percent of cells exhibiting membrane fluorescence was assessed using a Zeiss Orthoplan fluorescent microscope.
Effect of drugs on EAC3 rosette
formation
I, Th, CT, PGE,, PGE2, PGF,,, A, and NA. Fresh serial lOO-fold dilutions of these agents in phosphate-buffered saline were prepared for each experiment and incubated with lymphocytes as described. Methylprednisolone or propranolol. Lymphocytes were preincubated for 15 min with methylprednisolone (10m3 M
CLIN. IMMUNOL. SEPTEMBER 1979
to 10e7 M) or propranolol (lo-” M to 10 -I’ M) prior to their incubation with I, Th, or A. Puromycin. PU was added to the cell suspensions 15 min prior to adding I or Th and allowed to remain in the culture medium until all drugs were washed away just prior to EAC3 rosetting.
lmmunosorbent
column
preparation
Rabbit anti-human Fab was purified over a Sepharose 4B immunoabsorbent column containing human gamma globulin purified from normal serum by diethylaminoethyl cellulose chromatography. The antibody was eluted with 3 M sodium thiocyanate and dialyzed extensively against 0.1 M sodium bicarbonate prior to cross-linking to Sephadex G-200 beads. Sephadex G-200 was sieved to achieve uniform bead size (88 to 120 p), swollen in water, and activated with cyanogen bromide.” The activated Sephadex was gently mixed with purified rabbit antibody for 4 hr and then exhaustively washed. Approximately 0.25 to 0.35 mg protein was bound per milliliter of activated Sephadex. Disposable lo-ml syringes were fitted with polyethylene disks (Bell-Art Products, Panawock, N. J.) and packed with 5 ml Sephadex conjugate. IR The column was washed with HBSS-FCS prior to use.
Cell separation
studies
Lymphocytes were resuspended at a concentration of 20 x lo6 cells/ml in cold RPMI-1640 containing 10% FCS, calcium EDTA (0.092%), and 0.12 M HEPES and drops were applied to the 5-ml Sephadex anti-human Fab column. Nonretained cells were collected by stepwise elution with 15-ml aliquots of starting medium at a flow rate of 0.5 ml/min until the effluent was virtually cell free. Bound cells were recovered by gentle agitation and elution with two 15ml aliquots of medium containing 10 mg/ml human gamma globulin. The resulting cell suspension was enriched in B cells; 67% were EAC3 rosette-forming cells (EAC3RFC) while only 6% were E rosette- forming cells (E-RFC). The nonretained population was further fractionated into E rosette-positive and E rosette-negative cells. in brief, 10 ml lymphocyte suspension (4 x 10” cells/ml HBSS) were incubated with 1 ml 5% E and 1.5 ml FCS for 10 min at 37” C in an atmosphere of 5% COB in air. Cells were pelleted at 200 x g for 5 min in a 4” C centrifuge and incubated at 4” C for 1 hr. The pellet was gently resuspended, layered over a Ficoll-Hypaque gradient, and centrifuged at 400 x g for 30 min at room temperature. Cells that floated at the top of the gradient were designated null cells and contained less than 5% E-RFC and 20% to 30% EAC3-RFC. Cells that sedimented to the bottom of the gradient together with the sheep red cells were resuspended in 5 ml 0.87% ammonium chloride solution for 5 min at 37” C to lyse the red cells, and then were immediately washed with HBSS-FCS. The resulting lymphocyte suspension was designated T cells and contained more than 90% E-RFC and less than 6% EAC3-RFC. Unfractionated cells, T, B, and null cells were washed three times with HBSS-FCS prior to rosetting with EAC3.
VOLUME NUMBER
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64 3
RESULTS Effect of I and Th on EAC3 rosette
effects
on EAC3 rosettes
185
formation
Baseline EAC3 rosette formation was similar for all groups (Table I). Preincubation of normal peripheral blood lymphocytes (PBL) with I and Th for 1 hr resulted in a statistically significant increase in the percent EAC-RFC (Table I and Fig. 1). Mean increase of EAC3-RFC averaged 49% with I and 47% with Th for the two control groups of healthy subjects. Identical results were obtained when the incubation time of lymphocytes with these drugs was varied from a minimum 15 min to a maximum 3 hr. Maximal increase in EAC3-RFC occurred at a concentratitn of lo-” M I, IO-” M Th, and 1 rig/ml CT (Fig. 2, u-c). In contrast, EAC3 rosette formation by lymphocytes from patients with asthma was not significantly affected by preincubation with I, Th, or CT in doses covering the range of lop3 M to lo-l5 M (data not shown). Response of lymphocytes from 10 patients with allergic rhinitis was similar to that of normal lymphocytes, with mean increases in EAC3-RFC of 50% and 52%, following incubation with I and Th, respectively. The observed increase in the percent of normal EAC-RFC following incubation with I and Th reflects a true increase in the number of complement receptor-bearing lymphocytes, since cell counts and cell viability (by trypan blue exclusion) were identical for control and drug-treated cell suspensions of both normal and asthmatic subjects. Increases in EAC3 rosette formation by PBL were unaffected by treatment of normal donors with Th (1 gm daily for 2 days) or by overnight incubation of normal cells in serum (at a final concentration of 10% in RPMI-1640 medium) obtained from patients with asthma. Preincubation of lymphocytes from asthmatic subjects with methylprednisolone ( 10m3M to 10e7 M for 15 min) did not reverse the resistance of these lymphocytes to the enhancing effect of I and Th on EAC3 rosette formation (data not shown). Effect of fetal calf serum FCS from Grand Island Biological Co., Lots A475416 and A272325, was used throughout the present study. In preliminary studies it was found that in the presence of two other lots of FCS, G.I.B.CO. A775524 and A979117, EAC3 rosette formation by normal lymphocytes was not enhanced by I (mean change in nine experiments, + 1.1% whereas EAC3 rosette formation by lymphocytes from patients with asthma was decreased (mean change in nine experiments, -4.8%. In order to best study the effects of various agents that increase intracellular CAMP on
5?----3 5 7
9
II 13 I5
-
CONCENTRATION
-0 lOXMOLAR
FIG. 2. Dose-response effect of agents that tracellular CAMP levels on EAC3 rosette normal lymphocytes. Percent EAC3 rosette shown on the ordinate. Drug concentration the abscissa. Results represent mean k SD imentsa, Isoproterenol; b, theophylline; c, d, PGE,; e, adrenaline; t noradrenalin.
modulate information by formation is is shown on of five expercholera toxin;
EAC3 rosette formation in normal lymphocytes, FCS from lots supporting the I-mediated increase in normal EAC-RFC was used. Dialysis and heat treatment (60” C for 1 hr) of FCS did not affect its permissive effect on I-mediated enhancement of EAC3 rosette formation. Response of normal lymphocytes that modulate cAMP levels
to drugs
I, Th, CT, PGEl, and A all caused increases in EAC3 rosette formation by normal lymphocytes (Fig. 2, u-e). Propranolol, PGEz, and PGFz, did not affect EAC3 rosette formation (data not shown). Noradrenalin produced inhibition of EAC3 rosette formation (Fig. 2,f). This inhibition was not augmented by the addition of propranolol. Propranolol partially blocked the enhancing effects of I and A but not those of Th (Fig. 3, a-c). Effect of puromycin on l-mediated enhancement of EAC3 rosette formation Preincubation of lymphocytes with PU blocked I-mediated enhancement of EAC3 rosette formation. This effect was partial at a PU concentration of 1 pg/ml and complete at 10 ,ug/ml (Fig. 4). Response
of E-WC to I
Preincubation with I did not cause a change in the percent of E-RFC of lymphocytes from eight normal (before, 53.9 t 8.9; after, 54.7 * 8.8) and eight asthmatic (before, 57.6 ? 6.9; after, 56.7 + 6.8) subjects.
186
J. ALLERGY
Kohn et al.
CLIN. IMMUNOL. SEPTEMBER 1979
70s
f
-9 b, ADRENALINE lo-% MI OL---L---L--0 35791113
0
CONCENTRATION
357911
c 1 THEOPHYLLINE l&4
0
357911
OF PROPANOLOL IO-’ MOLAR
FIG. 3. Effect of propranolol on the enhancement of EAC3 rosette formation by (al IO-” M isoproterenol; lb) 1O-9 M adrenaline, and (cl 10m9 M theophylline. Percent EAC3 rosette formation is shown in the ordinates. Concentrations of propranolol are shown on the abscissa. The * represents the baseline value of EACB-RFC in the absence of all drugs.
W
‘;----I.,,, F
to-
a
5
El
T
IB 9
CONTROLS
I HULL I
-B I B I “NFRACTIONATED NULL I t ASTHMATIC
FIG. 5. Enhancement of EAC3 rosette formation by I in lymphocyte subpopulations of normal and asthmatic subjects. Lymphocyte subpopulations were incubated with I (10m9 M) for 1 hr prior to the EAC3 rosette assay. Percent EAC3 rosette formation is shown on the ordinate.
DISCUSSION
15.
l5 o
IB
following incubation of either unfractionated or null cells from control subjects.
w” 25. L ii 20B s a
-~-B IB LIHPnOCITE “NFRACPOPULPlTlONTIONATED I
---++
M X----X e----e
CONCENTRATION
ISOPROTERENOL ISOPROTERENOL+PUROMYCIN ( Irq/ml) ISOPROTERENOL+ PUROMYCIN (Ioyg/ml) OF ISOPROTERENOL
16” MOLAR
FIG. 4. Effect of puromycin on l-mediated enhancement of EAC3 rosette formation by normal lymphocytes. Percent EAC3 rosette formation is shown on the ordinate. Concentration of I is shown on the abscissa. Results represent mean -C SD of four experiments. The A represents the baseline percent EAC-RFC in the absence of all drugs.
Enhancement of EAC3 rosette I in lymphocyte subpopulations
formation
by
T cell-rich and B cell-rich subpopulations from normal individuals failed to demonstrate a significant increase in EAC3-RFC following preincubation with I; on the other hand the null cell population demonstrated a 50% increase (Fig. 5). Neither unfractionated nor null cell populations from asthmatic patients responded to I. Effect of I and Th on expression membrane immunoglobulins
of
No increase in the number of surface immunofluorescent staining cells was seen with I or Th
Expression of a surface receptor for the third component of complement (C3) is a characteristic of most of the B-lymphocytes as well as of 20% to 30% of the null lymphocytes. lg Modulation of C3 receptor expression by adrenergic agents has recently been reported by Ito et al. l5 who demonstrated that beta adrenergic agents increased EAC3 rosette formation in mouse spleen cells. The present article describes the enhancement of EAC3 rosette formation in normal human PBL by agents that increase intracellular CAMP, and demonstrates the lack of this enhancement in lymphocytes from subjects with asthma. Early in our studies of normal lymphocytes it became clear that only certain lots of FCS permitted I-mediated enhancement of EAC3 rosette formation. In the presence of other lots of FCS, EAC3 rosette formation was not affected by the addition of I. These results suggested the presence of a serum factor in certain lots of FCS which exerts a permissive effect on I-mediated increases in EAC3 rosette formation. This factor was demonstrated to be heat stable and nondialyzable (> 10,000 mw) and thus it is unlikely to be a prostaglandin. Similar variability in the capacity of different lots of FCS to support in vitro immune responses has been well documented.” To best examine the EAC3 response of normal lymphocytes to various agents that increase intracellular CAMP, we used only those lots of FCS which supported the l-mediated enhancement of normal lymphocyte EAC3 rosette formation.
VOLUME NUMBER
64 3
The beta adrenergic agent isoproterenol significantly increased EAC3-RFC in normal lymphocytes (Fig. 1, Table I) with an optimum concentration of lo-” M (Fig. 2, a). Lymphocytes from asthmatic subjects failed to show an increase after incubation with a broad range (lo-” M to 10-l” M) of I concentrations. Similar findings were observed with Th (Fig. 2, b, and Table I), CT, PGEl, and A (Fig. 2). The beta adrenergic antagonist propranolol blocked the enhancement of EAC-RFC induced by I and A (Fig. 3, a and h) but did not affect the enhancement of EAC3RFC induced by Th (Fig. 3, L.). All pharmacologic agents that caused enhancement of EAC3 rosette formation are known to cause an increase in intracellular CAMP.“” ” Intracellular CAMP levels were not measured. However, the present data suggest that the observed enhancement of EAC3 rosette formation is mediated through an increase in intracellular CAMP and/or through CAMP-triggered events and not solely through selective triggering of beta adrenergic receptors. The observed enhancement of EAC3 rosette formation in normal lymphocytes reflects an increase in the actual number of lymphocytes expressing complement receptors, since cell counts and viability were identical in control and in drug-incubated cell suspensions and since macrophages had been removed by overnight incubation of cells in plastic flasks. Furthermore, the data suggests that the increase in complement receptor- bearing lymphocytes is an active expression of new receptors and not merely an uncovering of already present receptors. Puromycin did not alter baseline EAC3 rosette formation but blocked I-mediated enhancement (Fig. 4) of EAC3-RFC, suggesting that de novo protein synthesis is required for the expression of new lymphocyte complement receptors. Cells expressing new EAC3 receptors were demonstrated to reside in the null cell fraction (Fig. 5). Such cells could not be detected in the null cell fraction from asthmatic patients. Expression of new EAC3-EFC was complete within 15 mitt, was stable for more than 3 hr, and was not accompanied by an increase in surface immunoglobulin-bearing cells . Our data demonstrate a fundamental difference between the response of normal lymphocytes and lymphocytes from asthmatics to I in the EAC3 rosetteforming assay regardless of the source of FCS. When permissive lots of FCS were used in the assay, I enhanced EAC3 rosette formation by lymphocytes from normal subjects and did not affect EAC3 rosette formation by lymphocytes from asthmatic patients over the entire dose range used ( 10e3M to lo-l5 M). When
Pharmacologic
effects
on EAC3 rosettes
187
nonpermissive lots of FCS were used, addition of I did not affect EAC3 rosette formation by normal lymphocytes and decreased the percent of EAC-RFC in lymphocytes from asthmatics. Several investigators 2, 5, 6. IO. 23-26 have o,,served that a variety of cells from patients with asthma demonstrate an altered sensitivity to beta adrenergic agents. This altered sensitivity of lymphocytes from asthmatics has been ascribed to beta adrenergic blockade, a theory first proposed by Szentivanyi.’ Thus Parker et al.“, 6, 26 and Logsdon et al.” have reported that leukocytes and lymphocytes from patients with asthma produce less CAMP than normal leukocytes and lymphocytes following incubation with catecholamines, and further demonstrated that the altered response of these cells can be reversed by in vivo3, 26or in vitro26 corticosteroid administration. On the other hand, other investigators have observed a diminished CAMP response of lymphocytes from asthmatics only during active bronchospasm.4. 9. l2 Conolly and Greenacre” and Morris et al.’ presented evidence that this diminished response was due to ongoing therapy with beta adrenergic agents and was not reversed by corticosteroids administered in viva.’ To control for all these factors, asthmatic patients included in the present study had normal lung function at the time of testing. Twenty-five of the 35 asthmatics had not received any beta adrenergic agents within 6 wk of the study, including seven patients who were receiving no medications at all and three patients who had had no bronchospasm or bronchodilator therapy for more than 5 yr. The remaining 10 asthmatic patients were receiving daily theophylline and metaproterenol and six of them were also receiving alternate-day corticosteroid therapy. In no case did medications affect the resistance of lymphocytes from patients with asthma to the enhancing effect of I, Th, and CT on EAC3 rosette formation; and lymphocytes from asthmatics continued to demonstrate resistance to the enhancement of EAC3 rosette formation following in vitro preincubation with methylprednisolone. Furthermore, I- and Th-mediated enhancement of EAC3 rosette formation in normal lymphocytes was unaffected by pretreatment of normal donors with theophylline. Lymphocytes from asthmatic patients also failed to respond to agents that increase intracellular CAMP by Bechanisms other than stimulation of the lymphocyte beta receptor. Thus, while normal lymphocytes responded to Th, CT, and PGEl with an increase in EAC3 rosette formation of a magnitude similar to that caused by I, lymphocytes from asthmatic subjects were totally resistant to the EAC3-enhancing effect of
188
Kohn et al.
J. ALLERGY
these agents. Furthermore, it was demonstrated that this resistance was intrinsic to these lymphocytes since incubation of normal lymphocytes with sera from asthmatic subjects did not affect the response of the normal cells. Since intracellular CAMP was not measured, the present findings suggest but do not conclusively prove that the CAMP response of the lymphocytes to the pharmacologic agents is abnormal in asthma. The differential response of lymphocytes from normal and asthmatic subjects to I and Th can be used as an adjunct in the investigation of patients with reversible airway disease. Studies of infants who are at risk of developing asthma (patients with bronchiolitis, offspring of asthmatic parents) are currently underway to determine if the lymphocyte abnormality described in the present report precedes the development of asthma. The authors are grateful to Dr. Fred Rosenfor reviewing the manuscript, Mr. Scott Ames and Ms. Lilya Naiman for technical assistance,and Miss Cathy Clark for secretarial assistance. REFERENCES 1. Szentivanyi A: The beta-adrenergic theory of atopic abnormality in bronchial asthma. J ALLERGY CLIN IMMUNOL 42~203, 1968. 2. Smith JW, Steiner AL, Newberry WM, Parker CW: Cyclic adenosine 3’) 5’-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation. J Clin Invest 50~432, 1971. 3. Logsdon PJ, Middleton E Jr, Coffey RG: Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and nonasthmatic subjects. J ALLERGY CLIN IMMUNOL 50~45,
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