Brief communication
Histamine receptor-bearing T lymphocytes in patients H. Verhaegen,
peripheral with allergies
M.D., W. De Cock, M.Sc., and J. De Cree, M.D.
Merksem (Antwerp),
Belgium
The effect of histamine on the capacity of T lymphocytes to form E rosettes was tested in 10 healthy subjects and in 13 patients with allergies. Histamine had no effect on the capacity of T lymphocytes to form E rosettes in healthy subjects, but signijicantly inhibited the E rosette ,formation of T lymphocytes in patients with allergies.
In the past two years there is increasing evidence that receptors for histamine, /l-adrenergic catecholamines, and prostaglandins E exist on animal lymphocytes under certain experimental conditions.1-4 These agents increase the intracellular level of cyclic adenosine monophosphate (CAMP) by stimulation of adenylate cyclase.3 Agents that increase intracellular CAMP are reported to inhibit the capacity of human T lymphocytes to form E rosettes53 6 We therefore studied the effect of histamine on the capacity of peripheral T lymphocytes to form E rosettes in healthy subjects and in patients with allergies. MATERIAL
AND METHODS
Venous blood was collected on heparin from 10 adult healthy subjects and from 13 patients with allergies, aged 1% to 62 yr (median, 24 yr), who were in an allergy-free period and were not taking medication. The patients were diagnosed as having hay fever (5), penicillin allergy (3), ampicillin allergy (l), shellfish allergy (I), and atopic dermatitis with elevated serum IgE (3).
Separation
of lymphocytes
A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml blood and allowing the erythrocytes
to sediment at room temperature
for about 1 hr.
The leukocyte-rich plasma was then layered onto a Ficoll/ Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1,500 rpm for 30 min. The lymphocytes at the interface were aspirated and washed with Hanks balanced salt solution (HBSS, pH 7.7), and the concentration was adjusted to 2.106 cells/ml in HBSS. Viability of the lymphocytes was tested with 1% tryptan blue, and purity was evaluated on a smear stained by the May-GriinwaldGiemsa method.
Sheep red blood cells (SRBC) SRBC were always obtained under sterile conditions in Alsever’s solution (v/v, L/L) from the same sheep. The cells were stored at 4” C and washed three times with HBSS before use. A 1% solution of SRBC in HBSS was prepared.
Experimental
Lymphocyte suspension, 0.2 ml, was mixed with 0.4 ml HBSS or 0.2 ml HBSS and 0.2 ml histamine HCl ( 10m6M to 10m3M) and incubated for 30 min at 37” C. SRBC suspension, 0.25 ml, was added to 0.25 ml of these lymphocyte suspensions, incubated for 5 min at 37” C, centrifuged at 700 rpm for 5 min, and then incubated for 1 hr at 0” C.’ After gentle resuspension, rosette-forming cells were counted in a hemocytometer. Two hundred cells were counted, and lymphocytes binding three or more SRBC were considered positive.
Statistical From the Clinical Research Unit, St. Bartholomeus Hospital. Received for publication June 7, 1976. Accepted for publication July 1, 1976. Reprint requests to: H. Verhaegen, M.D., Clinical Research Unit, St. Bartholomeus Hospital, B-2060 Merksem (Antwerp), Belgium.
Vol. 59, No. 3, pp. 266-268
design
analysis
Student’s t test, two-tailed,*
was used.
*Davies, 0. L.: Statistical methods in research and production, London, 1947, Oliver & Boyd, Ltd.
VOLUME NUMBER
Histamine
59 3
Healthy
receptor-bearing
peripheral
Allergic
subjects
T lymphocytes
267
patients
2 60=: ti e $ 500
LOP=
30 '
N.S.
N.S.
I
I 0
N.S.
I
3x10-6M 3~10-~M
N.S
3x10~$4
3~10-~M
0.016
0.009 0.005 0.002
I
I
I 0
3x10-% 3~10-~M
3x10-‘M
3~10-~M Histamine
FIG. ‘I. The effect of different concentrations of histamine in 5 healthy subjects and in 5 patients with allergies.
RESULTS As shown in Fig. 1, the incubation of peripheral lymphocytes with histamine at concentrations of 3. 1O-7 M up to 3. low4 M for 30 min at 37” C had no effect on the E rosette formation of T lymphocytes of healthy subjects. On the contrary, the same concentrations of histamine significantly inhibited the E rosette formation of T lymphocytes in patients with allergies. The maximal inhibitory effect of histamine was obtained at 3. lo-” M, and this concentration did not affect the viability of the lymphocytes. Overall results with 3.10m4 M histamine in 10 healthy subjects and in 13 patients with allergies are represented in Fig. 2. DISCUSSION Our results suggest the presence of an unusually high number of histamine receptors on a subpopulation of T lymphocytes in patients with allergies. Their detection was obtained by an indirect method, namely, the inhibition of E rosette formation of T lymphocytes by histamine. This inhibitory effect of histamine on T lymphocytes is possibly mediated by intracellular CAMP. Indeed, histamine increases intracellular CAMP levels of various cell types,3 and the increase
on the capacity
of T lymphocytes
HCL
to form E rosettes
of CAMP levels of T lymphocytes inhibits their capacity to form E rosettes. 5, 6 However, further studies are needed to substantiate this speculation. These histamine receptors on lymphocytes have already been demonstrated in animals under certain experimental conditions. Plaut, Lichtenstein, and Henney’ showed that mice immunized with alloantigen developed cytolytic T lymphocytes that contained histamine type 2 receptors4 Melmon and co-workers* immunized mice with SRBC and found that cells actively producing antibodies possessed receptors for histamine and other hormones. The same group showed that precursors of antibody-producing cells did not have such receptors, but in transfer experiments they found a subpopulation of suppressor T lymphocytes bearing histamine receptors. These animal experiments show that histamine receptors are not present in random fashion on all lymphocytes but develop concomitantly with the commitment of a clone of immunocompetent cells. The histamine receptors we found on some of the T lymphocytes of patients with allergies could be such a subpopulation of immunocompetent cells acquired by multiple contacts with the allergen. However, the physiologic meaning of these histamine-bearing T
266
Verhaegen,
De Cock, and De Cree
Healthy
J. ALLERGY
subjects
Allergic
patients
0 A m X
6( I-
CLIN.
IMMUNOL. MARCH 1977
Hay fever Drug allergy Atopic dermatitis Food allergy
7cI-
)-
l-
LO,-
I3:: 30l-
N.S.
I
Control
I
3x10-‘M Histamine
c 0.001
I
Control
1
3 x~O-~M Histamine
FIG. 2. The effect of histamine on the capacity of peripheral T lymphocytes to form E rosettes in 10 healthy subjects and in 13 patients with allergies. Open symbols represent a duplicate determination on a different day.
lymphocytes in patients with allergies remains unclear-are they effector cells actively involved in the inflammatory reaction or are they regulatory cells suppressing, the inflammatory reaction caused by allergen IgE-mediated release of histamine and other mediators from mast cells and basophils? REFERENCES 1. Plaut, M., Lichtenstein, L. M., and Henney, C. S.: Increase in histamine receptors on thymus-derived effector lymphocytes during the primary immune response to alloantigens, Nature (Lond.) 244~284, 1973. 2. Melmon, K. L., Bourne, H. R., Weinstein, Y., Shearer, G. H., Bauminger, S., and Kram, J.: Hemolytic plague formation by leukocytes in vitro. Control by vasoactive hormones, J. Clin. Invest. 53~13, 1974.
3. Bourne, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer, G. H.: Modulation of inflammation and immunity by cyclic AMP, Science 184:19, 1974. 4. Plaut, M., Lichtenstein, L. M., and Henney, C. S.: Properties of a subpopulation of T-cells bearing histamine receptors, J. Clin. Invest. 55:856, 1975. 5. Chisari, F. V., and Edgington, T. S.: Human T-lymphocyte E-rosette function. I. A process modulated by intracellular cyclic AMP, J. Exp. Med. 140:1122, 1974. 6. Galant, S. P., and Remo, R. A.: P-adrenergic inhibition of human T-lymphocyte rosette, J. Immunol. 114~512, 1975. 7. Jondal, M., Helm, G., and Wigzell, H.: Surface markers on human T- and B-lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells, J. Exp. Med. 1X207, 1972.
Histamine receptor-bearing T lymphocytes in patients H. Verhaegen,
peripheral with allergies
M.D., W. De Cock, M.Sc., and J. De Cree, M.D.
Merksem (Antwerp),
Belgium
The effect of histamine on the capacity of T lymphocytes to form E rosettes was tested in 10 healthy subjects and in 13 patients with allergies. Histamine had no effect on the capacity of T lymphocytes to form E rosettes in healthy subjects, but signijicantly inhibited the E rosette ,formation of T lymphocytes in patients with allergies.
In the past two years there is increasing evidence that receptors for histamine, /l-adrenergic catecholamines, and prostaglandins E exist on animal lymphocytes under certain experimental conditions.1-4 These agents increase the intracellular level of cyclic adenosine monophosphate (CAMP) by stimulation of adenylate cyclase.3 Agents that increase intracellular CAMP are reported to inhibit the capacity of human T lymphocytes to form E rosettes53 6 We therefore studied the effect of histamine on the capacity of peripheral T lymphocytes to form E rosettes in healthy subjects and in patients with allergies. MATERIAL
AND METHODS
Venous blood was collected on heparin from 10 adult healthy subjects and from 13 patients with allergies, aged 1% to 62 yr (median, 24 yr), who were in an allergy-free period and were not taking medication. The patients were diagnosed as having hay fever (5), penicillin allergy (3), ampicillin allergy (l), shellfish allergy (I), and atopic dermatitis with elevated serum IgE (3).
Separation
of lymphocytes
A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml blood and allowing the erythrocytes
to sediment at room temperature
for about 1 hr.
The leukocyte-rich plasma was then layered onto a Ficoll/ Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1,500 rpm for 30 min. The lymphocytes at the interface were aspirated and washed with Hanks balanced salt solution (HBSS, pH 7.7), and the concentration was adjusted to 2.106 cells/ml in HBSS. Viability of the lymphocytes was tested with 1% tryptan blue, and purity was evaluated on a smear stained by the May-GriinwaldGiemsa method.
Sheep red blood cells (SRBC) SRBC were always obtained under sterile conditions in Alsever’s solution (v/v, L/L) from the same sheep. The cells were stored at 4” C and washed three times with HBSS before use. A 1% solution of SRBC in HBSS was prepared.
Experimental
Lymphocyte suspension, 0.2 ml, was mixed with 0.4 ml HBSS or 0.2 ml HBSS and 0.2 ml histamine HCl ( 10m6M to 10m3M) and incubated for 30 min at 37” C. SRBC suspension, 0.25 ml, was added to 0.25 ml of these lymphocyte suspensions, incubated for 5 min at 37” C, centrifuged at 700 rpm for 5 min, and then incubated for 1 hr at 0” C.’ After gentle resuspension, rosette-forming cells were counted in a hemocytometer. Two hundred cells were counted, and lymphocytes binding three or more SRBC were considered positive.
Statistical From the Clinical Research Unit, St. Bartholomeus Hospital. Received for publication June 7, 1976. Accepted for publication July 1, 1976. Reprint requests to: H. Verhaegen, M.D., Clinical Research Unit, St. Bartholomeus Hospital, B-2060 Merksem (Antwerp), Belgium.
Vol. 59, No. 3, pp. 266-268
design
analysis
Student’s t test, two-tailed,*
was used.
*Davies, 0. L.: Statistical methods in research and production, London, 1947, Oliver & Boyd, Ltd.
VOLUME NUMBER
Histamine
59 3
Healthy
receptor-bearing
peripheral
Allergic
subjects
T lymphocytes
267
patients
2 60=: ti e $ 500
LOP=
30 '
N.S.
N.S.
I
I 0
N.S.
I
3x10-6M 3~10-~M
N.S
3x10~$4
3~10-~M
0.016
0.009 0.005 0.002
I
I
I 0
3x10-% 3~10-~M
3x10-‘M
3~10-~M Histamine
FIG. ‘I. The effect of different concentrations of histamine in 5 healthy subjects and in 5 patients with allergies.
RESULTS As shown in Fig. 1, the incubation of peripheral lymphocytes with histamine at concentrations of 3. 1O-7 M up to 3. low4 M for 30 min at 37” C had no effect on the E rosette formation of T lymphocytes of healthy subjects. On the contrary, the same concentrations of histamine significantly inhibited the E rosette formation of T lymphocytes in patients with allergies. The maximal inhibitory effect of histamine was obtained at 3. lo-” M, and this concentration did not affect the viability of the lymphocytes. Overall results with 3.10m4 M histamine in 10 healthy subjects and in 13 patients with allergies are represented in Fig. 2. DISCUSSION Our results suggest the presence of an unusually high number of histamine receptors on a subpopulation of T lymphocytes in patients with allergies. Their detection was obtained by an indirect method, namely, the inhibition of E rosette formation of T lymphocytes by histamine. This inhibitory effect of histamine on T lymphocytes is possibly mediated by intracellular CAMP. Indeed, histamine increases intracellular CAMP levels of various cell types,3 and the increase
on the capacity
of T lymphocytes
HCL
to form E rosettes
of CAMP levels of T lymphocytes inhibits their capacity to form E rosettes. 5, 6 However, further studies are needed to substantiate this speculation. These histamine receptors on lymphocytes have already been demonstrated in animals under certain experimental conditions. Plaut, Lichtenstein, and Henney’ showed that mice immunized with alloantigen developed cytolytic T lymphocytes that contained histamine type 2 receptors4 Melmon and co-workers* immunized mice with SRBC and found that cells actively producing antibodies possessed receptors for histamine and other hormones. The same group showed that precursors of antibody-producing cells did not have such receptors, but in transfer experiments they found a subpopulation of suppressor T lymphocytes bearing histamine receptors. These animal experiments show that histamine receptors are not present in random fashion on all lymphocytes but develop concomitantly with the commitment of a clone of immunocompetent cells. The histamine receptors we found on some of the T lymphocytes of patients with allergies could be such a subpopulation of immunocompetent cells acquired by multiple contacts with the allergen. However, the physiologic meaning of these histamine-bearing T
266
Verhaegen,
De Cock, and De Cree
Healthy
J. ALLERGY
subjects
Allergic
patients
0 A m X
6( I-
CLIN.
IMMUNOL. MARCH 1977
Hay fever Drug allergy Atopic dermatitis Food allergy
7cI-
)-
l-
LO,-
I3:: 30l-
N.S.
I
Control
I
3x10-‘M Histamine
c 0.001
I
Control
1
3 x~O-~M Histamine
FIG. 2. The effect of histamine on the capacity of peripheral T lymphocytes to form E rosettes in 10 healthy subjects and in 13 patients with allergies. Open symbols represent a duplicate determination on a different day.
lymphocytes in patients with allergies remains unclear-are they effector cells actively involved in the inflammatory reaction or are they regulatory cells suppressing, the inflammatory reaction caused by allergen IgE-mediated release of histamine and other mediators from mast cells and basophils? REFERENCES 1. Plaut, M., Lichtenstein, L. M., and Henney, C. S.: Increase in histamine receptors on thymus-derived effector lymphocytes during the primary immune response to alloantigens, Nature (Lond.) 244~284, 1973. 2. Melmon, K. L., Bourne, H. R., Weinstein, Y., Shearer, G. H., Bauminger, S., and Kram, J.: Hemolytic plague formation by leukocytes in vitro. Control by vasoactive hormones, J. Clin. Invest. 53~13, 1974.
3. Bourne, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Weinstein, Y., and Shearer, G. H.: Modulation of inflammation and immunity by cyclic AMP, Science 184:19, 1974. 4. Plaut, M., Lichtenstein, L. M., and Henney, C. S.: Properties of a subpopulation of T-cells bearing histamine receptors, J. Clin. Invest. 55:856, 1975. 5. Chisari, F. V., and Edgington, T. S.: Human T-lymphocyte E-rosette function. I. A process modulated by intracellular cyclic AMP, J. Exp. Med. 140:1122, 1974. 6. Galant, S. P., and Remo, R. A.: P-adrenergic inhibition of human T-lymphocyte rosette, J. Immunol. 114~512, 1975. 7. Jondal, M., Helm, G., and Wigzell, H.: Surface markers on human T- and B-lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells, J. Exp. Med. 1X207, 1972.
