Con Jnctival hyperresponsiveness to ocular hist-maine challenge in patients with vernal conjunctivitis Stefano Bonini, MD,* Sergio Bonini, MD,** Mauro Schiavone, MD,* Marco Centofanti, MD,* Mathea R. Ailansmith, MD,*** and Massimo G. Bocci, MD* Rome, Italy, and Boston, Mass. We compared the conjunctival responsiveness to histamine diphosphate in patients with vernal conjunctivitis and in healthy control subjects. Fourteen asymptomatic patients with vernal conjunctivitis and 10 healthy volunteers were challenged in one eye with 10 tzl of increasing doses (0.01, 0.05, 0.5, and I mg/ml in phosphate-buffered saline)of histamine diphosphate. The contralateral eye was challenged with the diluent only. Photographs of each eye were taken for evaluation of conjunctival redness by two masked investigators. Both patients with vernal conjunctivitis and control subjects reacted to histamine with a dose-dependent conjunctival redness 2 to 5 minutes after ocular challenge. Conjunctival redness was more intense in patients with vernal conjunctivitis than in control subjects after ocular challenge with 0.01 and 0.05 mg / ml of histamine diphosphate solution (p < 0.05). Moreover, the threshoM concentration of histamine diphosphate, extrapolated from each individual dose-response curve, was significantly (p < 0.02) lower in patients with vernal conjunctivitis than in control subjects. Our findings suggest that patients with vernal conjunctivitis demonstrate conjunctival hyperresponsiveness t~ a nonspecific challenging agent. Nonspecific conjunctival hyperreactivity, a novel concept in allergic eve disease, may be relevant for a better understanding of the pathogenesis and clinical variability of vernal conjunctivitis. ( J ALL6RGY CLtN IMMUNOC 1992:89:103-7.) Key words: Vernal conjunctivitis, hyperreactivi~, ocular allergy
Extensive evidence indicates that allergic patients may be hyperreactive to various nonspecific substances in addition to the sensitizing allergens.1 Although close relationships exist between reactivity to allergens and to nonallergic substances, specific and nonspecific hyperreactivity represent distinct concurrent mechanisms in the pathogenesis of allergic diseases.2.3 Accordingly, the evaluation of nonspecific reactivity is an important factor in diagnosing and monitoring several atopic diseases, such as bronchial asthma.4. 5 Moreover, the treatment of nonspecific reactivity in the target organs is essential for successful therapy of allergic diseases. 6' 7 Fromthe *Departmentof Ophthalmology,Universityof Rome"Tor Vergata," the **Andrea Cesalpino Foundation, University of Rome "La Sapienza," and the ***EyeResearch Institute and Harvard Medical School, Boston, Mass. Received for publicationMay 7, 1991. Revised Aug. 6, 1991. Acceptedfor publication Aug. 26, 1991. Reprintrequests: StefanoBonini,MD, Via CassiaAntica35, 00191 Rome, Italy. 1/1/33368
Abbreviations used
PBS: Phosphate-buffered saline PC: Provoking concentration
Vernal conjunctivitis is a bilateral, inflammatory condition of the eye. It occurs in early spring, but symptoms may persist throughout the year. The clinical picture, the presence of eosinophils in the conjunctiva, and elevated levels of IgE in serum and tears all suggest the allergic nature of vernal conjunctivitis.8 However, the ocular manifestation cannot be explained by an immediate type 1 hypersensitivity mechanism alone. In fact, a history of other atopic disease is present in 15% to 65% o f patients with vernal conjunctivitis, and allergic sensitization is detectable by skin tests or RAST in only 54% of patients with vernal conjunctivitiis. 9-" In addition, the clinical variability of symptoms, even in the same season, strongly suggests that other factors, allergic or nonallergic, might also be involved in the pathogenesis of this disease.
103
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Bonini et al.
J. ALLERGYCLIN,IMMUNOL. JANUARY 1992
TABLE I. Characteristics and scores of patients and control subjects
Patient No.
6 7 8 9 10 11 12 13 14 Control subjects 1
2 3 4 5 6 7 8 9 10
Age (yr)
Sex
Age at onset (yr)
11 13 12 10 5 12 32
M M M M M F F
3 10 3 2 4 1 20
Clinical scores after histamine diphosphate (mg/ml) Skin prick test/RAST
11
F
7
10 8 10 9 11 9
M M M M M M
4 2 2 4 4 4
Neg Neg Neg Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg
20 22 30 18 30 34 23 22 22
F F M F F M F M M
----------
Neg Neg Neg Neg Neg Neg Neg Neg Neg
18
F
--
Neg
0.01
0.05
0.5
1
2 3 3 4
5 7 5 7
8 10 8 10
10 -10 --
1 1 1 1
3 3 3 4
8 7 8 8
10 10 9
2 2 5 4 5 3
6 5 9 6 9 5
10 8 -9 -7
10 -10 ---10
0 0 0
2 3 2
5 7 6
9 10 8
1
5
9
--
4
9
--
--
1
3
7
9
2 2 2 0
4 4 4 3
9 10 9 6
---8
Neg, Negative;pos, positive.
Histamine is a nonspecific challenging agent commonly used to evaluate nonspecific hyperresponsivehess in the airways and nasal mucosa. In the eye of the guinea pig, topical histamine challenge has been used to evaluate the conjunctival vascular permeability after an ocular immediate hypersensitivity reaction. 22 Ocular histamine challenge has also been used in humans to mimic signs and symptoms of ocular allergic diseases. ~3 To the best of our knowledge, histamine challenge has not previously been used as a parameter of nonspecific conjunctival response. In the present study, we investigated the degree of conjunctival responsiveness in patients with vernal conjunctivitis after increasing doses of histamine diphosphate topically applied to the eye and compared the responses with responses of nonallergic, healthy individuals. PATIENTS AND METHODS
Fourteen asymptomatic patients (11 male and three female patients) with a previous clinical diagnosis of vernal conjunctivitis were selected (Table I). Diagnosis based on
history, clinical examination, and the presence of eosinophils in the conjunctival scraping was made by one of us at least 1 year before the beginning of the study. Skin prick tests and/or RAST (Pharmacia, Uppsala, Sweden) to common allergens was also performed at that time to detect any IgE-mediated hypersensitivity. Seven patients were skin test and/or RAST negative, and seven were skin test and/or RAST positive (four to Dermatophagoides pteronyssinus, three to grass, two to Parietaria officinalis, and one patient to food allergens). Two patients had more than one sensitivity. Mean patient age was 11 years (range, 5 to 32 years). The study was performed out of the vernal season, and all patients had either no or mild ocular symptoms at the time of the study. Patients took no ocular or systemic medication for at least 1 month before entering the study. Ten healthy volunteers (four male and six female volunteers) with normal findings in eye examinations served as control subjects. Their mean age was 23 years (range, 18 to 34 years). All control subjects were skin test and/or RAST negative (Table I). All patients and control subjects were challenged with 10 ~1 of increasing doses of histamine diphosphate in the inferior conjunctival fornix of one eye. Doses of histamine diphosphate (Sigma Chemical Co., St. Louis, Mo.), 0.01,
VOLUME 89 NUMBER 1, PART 1
Hyperreactivity
in v e r n a l c o n i u n , ' l i v i t i s
105
_+0.8 _+0.2 10
_+1.8 +_1.1 9.4
+_1.9
CO 09 UJ Z rm LU ndE < _J
]-
7.8
+_2.0
+_t .4
L.)
3.9
O
i
/
_.L._
+_1.3
0.01
0.05
0.5
1.0
HISTAMINE DIPHOSPHATE mg/ml FIG. 1. Mean scores (+ SD) of ocular redness after increasing doses of histamine diphosphate in control subjects (open bars) and in patients with vernal conjunctivitis (hatched bars), The differences at 0.01 and 0.05 mg/ml are significant (p < 0.05),
0.05, 0.5, and 1 mg/ml in PBS solution, were administered at 15-minute intervals. The fellow eye was challenged with PBS at the same time intervals. Photographs were taken with an autoframe close-up camera (Lester Dine Inc., Farmingdale, N.Y.) before and within 5 minutes after each ocular challenge. Ocular redness on photographs was evaluated by two masked readers. Photographs were scored from 0 (no redness) to 3 + (maximum redness) for the inferior, nasal, and temporal bulbar conjunctiva, respectively (total maximum score, 9 + ). An intense redness of all three areas accompanied by a mild chemosis was scored as 10 + . Once a score of 9 + or 10 + was reached, the ocular challenge was stopped, and the subsequent clinical scores were arbitrarily evaluated as 10 + for statistical analysis. A two-tailed nonparametric statistical test (Mann-Whitney U) '~ was performed to analyze the results. A p < 0.05 was considered statistically significant.
RESULTS A dose-dependent increase in ocular redness was elicited by increasing doses of histamine diphosphate both in healthy control subjects and in patients with
vernal conjunctivitis (Table I). However, the response was more intense in the patients than in the conirol subjects, as was evident by the analysis of both mean and individual responses. The mean redness scores at the two lowest doses of histamine diphosphate (0.01 and 0.05 m g / m l ) were significantly higher ( p < 0.05) in patients w~th vernal conjunctivitis than in the control subjects (Fig, t). In addition, at 0.01 m g / m l of histamine diphosphate, the skin test- a n d / o r RAST-negative patients demonstrated significantly higher symptom scores than did the allergic (lgE positive) patients. To express quantitatively the degree o f individual reactivity, we calculated for each patient and control subject, a histamine diphosphate PC. This value was arbitrarily calculated, on the basis of the individual dose-response curves as the dose o f histamine diphosphate that induced a score equal to 2 SD over the mean score of healthy control subjects when they were challenged with a 0.01 m g / m l solution of histamine diphosphate. In our study, this score value wa~ 3.82.
106
Bonini et al.
J. ALLERGYCUN. IMMUNOL. JANUARY 1992
0.3-
0.2--
O0
O3
E LU F
Extensive evidence indicates that allergic patients may be hyperreactive to various nonspecific substances in addition to the sensitizing allergens.1 Although close relationships exist between reactivity to allergens and to nonallergic substances, specific and nonspecific hyperreactivity represent distinct concurrent mechanisms in the pathogenesis of allergic diseases.2.3 Accordingly, the evaluation of nonspecific reactivity is an important factor in diagnosing and monitoring several atopic diseases, such as bronchial asthma.4. 5 Moreover, the treatment of nonspecific reactivity in the target organs is essential for successful therapy of allergic diseases. 6' 7 Fromthe *Departmentof Ophthalmology,Universityof Rome"Tor Vergata," the **Andrea Cesalpino Foundation, University of Rome "La Sapienza," and the ***EyeResearch Institute and Harvard Medical School, Boston, Mass. Received for publicationMay 7, 1991. Revised Aug. 6, 1991. Acceptedfor publication Aug. 26, 1991. Reprintrequests: StefanoBonini,MD, Via CassiaAntica35, 00191 Rome, Italy. 1/1/33368
Abbreviations used
PBS: Phosphate-buffered saline PC: Provoking concentration
Vernal conjunctivitis is a bilateral, inflammatory condition of the eye. It occurs in early spring, but symptoms may persist throughout the year. The clinical picture, the presence of eosinophils in the conjunctiva, and elevated levels of IgE in serum and tears all suggest the allergic nature of vernal conjunctivitis.8 However, the ocular manifestation cannot be explained by an immediate type 1 hypersensitivity mechanism alone. In fact, a history of other atopic disease is present in 15% to 65% o f patients with vernal conjunctivitis, and allergic sensitization is detectable by skin tests or RAST in only 54% of patients with vernal conjunctivitiis. 9-" In addition, the clinical variability of symptoms, even in the same season, strongly suggests that other factors, allergic or nonallergic, might also be involved in the pathogenesis of this disease.
103
104
Bonini et al.
J. ALLERGYCLIN,IMMUNOL. JANUARY 1992
TABLE I. Characteristics and scores of patients and control subjects
Patient No.
6 7 8 9 10 11 12 13 14 Control subjects 1
2 3 4 5 6 7 8 9 10
Age (yr)
Sex
Age at onset (yr)
11 13 12 10 5 12 32
M M M M M F F
3 10 3 2 4 1 20
Clinical scores after histamine diphosphate (mg/ml) Skin prick test/RAST
11
F
7
10 8 10 9 11 9
M M M M M M
4 2 2 4 4 4
Neg Neg Neg Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg
20 22 30 18 30 34 23 22 22
F F M F F M F M M
----------
Neg Neg Neg Neg Neg Neg Neg Neg Neg
18
F
--
Neg
0.01
0.05
0.5
1
2 3 3 4
5 7 5 7
8 10 8 10
10 -10 --
1 1 1 1
3 3 3 4
8 7 8 8
10 10 9
2 2 5 4 5 3
6 5 9 6 9 5
10 8 -9 -7
10 -10 ---10
0 0 0
2 3 2
5 7 6
9 10 8
1
5
9
--
4
9
--
--
1
3
7
9
2 2 2 0
4 4 4 3
9 10 9 6
---8
Neg, Negative;pos, positive.
Histamine is a nonspecific challenging agent commonly used to evaluate nonspecific hyperresponsivehess in the airways and nasal mucosa. In the eye of the guinea pig, topical histamine challenge has been used to evaluate the conjunctival vascular permeability after an ocular immediate hypersensitivity reaction. 22 Ocular histamine challenge has also been used in humans to mimic signs and symptoms of ocular allergic diseases. ~3 To the best of our knowledge, histamine challenge has not previously been used as a parameter of nonspecific conjunctival response. In the present study, we investigated the degree of conjunctival responsiveness in patients with vernal conjunctivitis after increasing doses of histamine diphosphate topically applied to the eye and compared the responses with responses of nonallergic, healthy individuals. PATIENTS AND METHODS
Fourteen asymptomatic patients (11 male and three female patients) with a previous clinical diagnosis of vernal conjunctivitis were selected (Table I). Diagnosis based on
history, clinical examination, and the presence of eosinophils in the conjunctival scraping was made by one of us at least 1 year before the beginning of the study. Skin prick tests and/or RAST (Pharmacia, Uppsala, Sweden) to common allergens was also performed at that time to detect any IgE-mediated hypersensitivity. Seven patients were skin test and/or RAST negative, and seven were skin test and/or RAST positive (four to Dermatophagoides pteronyssinus, three to grass, two to Parietaria officinalis, and one patient to food allergens). Two patients had more than one sensitivity. Mean patient age was 11 years (range, 5 to 32 years). The study was performed out of the vernal season, and all patients had either no or mild ocular symptoms at the time of the study. Patients took no ocular or systemic medication for at least 1 month before entering the study. Ten healthy volunteers (four male and six female volunteers) with normal findings in eye examinations served as control subjects. Their mean age was 23 years (range, 18 to 34 years). All control subjects were skin test and/or RAST negative (Table I). All patients and control subjects were challenged with 10 ~1 of increasing doses of histamine diphosphate in the inferior conjunctival fornix of one eye. Doses of histamine diphosphate (Sigma Chemical Co., St. Louis, Mo.), 0.01,
VOLUME 89 NUMBER 1, PART 1
Hyperreactivity
in v e r n a l c o n i u n , ' l i v i t i s
105
_+0.8 _+0.2 10
_+1.8 +_1.1 9.4
+_1.9
CO 09 UJ Z rm LU ndE < _J
]-
7.8
+_2.0
+_t .4
L.)
3.9
O
i
/
_.L._
+_1.3
0.01
0.05
0.5
1.0
HISTAMINE DIPHOSPHATE mg/ml FIG. 1. Mean scores (+ SD) of ocular redness after increasing doses of histamine diphosphate in control subjects (open bars) and in patients with vernal conjunctivitis (hatched bars), The differences at 0.01 and 0.05 mg/ml are significant (p < 0.05),
0.05, 0.5, and 1 mg/ml in PBS solution, were administered at 15-minute intervals. The fellow eye was challenged with PBS at the same time intervals. Photographs were taken with an autoframe close-up camera (Lester Dine Inc., Farmingdale, N.Y.) before and within 5 minutes after each ocular challenge. Ocular redness on photographs was evaluated by two masked readers. Photographs were scored from 0 (no redness) to 3 + (maximum redness) for the inferior, nasal, and temporal bulbar conjunctiva, respectively (total maximum score, 9 + ). An intense redness of all three areas accompanied by a mild chemosis was scored as 10 + . Once a score of 9 + or 10 + was reached, the ocular challenge was stopped, and the subsequent clinical scores were arbitrarily evaluated as 10 + for statistical analysis. A two-tailed nonparametric statistical test (Mann-Whitney U) '~ was performed to analyze the results. A p < 0.05 was considered statistically significant.
RESULTS A dose-dependent increase in ocular redness was elicited by increasing doses of histamine diphosphate both in healthy control subjects and in patients with
vernal conjunctivitis (Table I). However, the response was more intense in the patients than in the conirol subjects, as was evident by the analysis of both mean and individual responses. The mean redness scores at the two lowest doses of histamine diphosphate (0.01 and 0.05 m g / m l ) were significantly higher ( p < 0.05) in patients w~th vernal conjunctivitis than in the control subjects (Fig, t). In addition, at 0.01 m g / m l of histamine diphosphate, the skin test- a n d / o r RAST-negative patients demonstrated significantly higher symptom scores than did the allergic (lgE positive) patients. To express quantitatively the degree o f individual reactivity, we calculated for each patient and control subject, a histamine diphosphate PC. This value was arbitrarily calculated, on the basis of the individual dose-response curves as the dose o f histamine diphosphate that induced a score equal to 2 SD over the mean score of healthy control subjects when they were challenged with a 0.01 m g / m l solution of histamine diphosphate. In our study, this score value wa~ 3.82.
106
Bonini et al.
J. ALLERGYCUN. IMMUNOL. JANUARY 1992
0.3-
0.2--
O0
O3
E LU F
