Int. Archs Allergy appl. Immun. 52: 48-52 (1976)
Factors in the Tissue Eosinophil Responses in Human Immediate Hypersensitivity Reactions Burton Zweiman, Robert I. Slott and Paul C. Atkins Allergy and Immunology Section, VA Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract. Factors underlying the eosinophil responses in the tissue sites of immediate hypersensitivity reactions have been investigated. A quantitative assessment of the number of eosinophils appearing in skin test reactions to ragweed antigen and compound 48/80 in rag weed-sensitive humans has been compared with several other parameters. There was a moderate, statistically significant, correlation with serum levels of IgE-class antiragweed antibody; also, eosinophil responses were minimal or absent in minimally positive threshold dilution skin tests. Tissue eosinophil responses were generally limited to those with baseline blood eosinophil levels of at least 150 mm3; however, there was no correlation between blood and tissue eosinophil levels in individual subjects. These findings suggest that the pathogenesis of the observed tissue eosinophil responses may be multifactorial.
Received: February 12, 1976.
carried out for several years sequential his tologic studies of cellular inflammatory res ponses in immediate hypersensitivity reac tions in human skin [3, 4]. The cellular res ponses we have seen are qualitatively simi lar to those present in many clinical allergic reactions. Recently, we have utilized modi fications permitting a more quantitative as sessment of the eosinophil response. It has, therefore, been feasible to compare the de gree of such responses in antigen-induced reactions with the degree of allergic reactivi ty of the host, the blood eosinophil level at the initiation of the reaction, and cellular Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:04:55 PM
Although it has long been known that prominent eosinophilic infiltration is com mon in tissue sites of human allergic reac tions, the factors underlying the degree of such eosinophilic responses in an individual patient are not well defined. Several lines of investigation in recent years have uncovered several factors with in vivo chemotactic ac tivity for eosinophils [1] with at least one factor likely of mast cell origin [2], Compa rable in vivo kinetic studies of allergic res ponses in the human respiratory mucosa have been limited by ethical and logistic considerations. For these reasons, we have
49
Zweiman/Slott/Atkins
Materials and Methods Patient Group 15 atopic subjects, ages 21-30, exhibiting strong wheal and flare reactions 20 min after intradermal testing with 20 PNU of mixed ragweed extract (prepared in the Allergy and Immunology Clinic, University of Pennsylvania) were studied, at times other than the ragweed pollinating sea son. No subjects were receiving symptomatic med ication or hyposensitization therapy at the time of study. Skin Test As described previously by us [4], threshold di lution skin testing with ragweed extract was car ried out in the lateral aspect of the upper arm, us ing intradermal injection of 0.02-ml aliquots con taining, respectively, 20, 6, 2, 0, 0.6, 0.2, 0.06, 0.02, 0.006, and 0.002 PNU. At another site, 0.02 of a 35 mg/ml solution of compound 48/80 (Burroughs-Wellcome, Research Triangle Park, N.C.) was injected intradermally. All gross reactions were read at 20 min by an individual unaware of the clinical history of the subjects. A reaction was considered positive if the mean wheal diameter was greater than 8 mm and at least twice that of the negative control.
found in these earlier studies that eosinophil res ponses were prominent by this time lasting for at least several hours thereafter. Biopsies were fixed in neutral bufferred formalin, sectioned at 6 pm and stained with May-Grunewald-Giemsa. Sec tions were coded and then overlaid with a translu cent nylon grid composed of squares 0.5 X 0.5 mm, permitting systematic counting of the eosino phils and mast cells in ten such grid squares (2.5 mm2) of dermis starting at the dermal-epidermal junction were recorded. Repeat readings of the same tissue section by the same observer showed good consistency with this method [4]. Selected biopsies were read independently by a second ob server as well, with good agreement between the independent readings. Statistical Analysis All the data from the different types of mea surement were tabulated separately at the conclu sion of the studies, before the codes on the serum specimens and tissue biopsies were broken. Corre lations were then attempted where appropriate, using a regression coefficient program on a Mon roe calculator.
Results
The total blood eosinophil levels, and tissue eosinophil responses/2.5 mm2 to the intradermal injection of ragweed both in the dose of 20 PNU and the extinction dilution are shown in table I. Also shown are the tis Blood Studies Immediately before the intradermal injection sue eosinophil responses to 48/80, and the of ragweed or compound 48/80, venous blood was tissue mast cell levels after injection of rag obtained for (1) total eosinophil count (counted weed and 48/80. It is apparent that there is with the Uno pipette system, Becton Dickinson considerable variation within the study Co.), and (2) serum specimens, coded and then an group of the tissue eosinophil responses to alyzed for antiantigen E antibodies of the IgE class by the Pharmacia Company, Piscataway, both ragweed and 48/80. Yet this was not N.J. simply a function of the blood eosinophil levels at the time of intradermal injections. Skin Biopsies It is true that tissue eosinophil responses 2 h after the injection, 3 mm diameter pinch biopsies were obtained at selected injection sites of more than 10 cells/2.5 mm2 were seen at ragweed extract, and compound 48/80, by tech ragweed 20 PNU skin test sites for the most part in those subjects with blood eosinophil niques previously described by us [3], We had
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:04:55 PM
responses in nonallergic reactions induced by compound 48/80. This report describes such comparative studies.
Zweiman/Slott/Atkins
50
Subject Blood eosino phil levels R.B. J.L. J.M. R.M. F.M. M.M. J.P. K.R. C.B. J.B. E.G. J.K. H.N. R.S. E.T.
193 320 306 288 384 400 176 256 158 80 144 96 112 144 112
Tissue eosino phil re sponse to ragweed 36 42 52 54 84 10 135 102 20 6 110 9 0 0 8
Tissue eosino phil re sponse to 48/80
Tissue mast cells
135 34 12 104 29 12 57 0 15 10 85 4 0 0 18
12 28 9 4 10 10 3 16 13 20 6 22 16 8 20
after after ragweed 48/80 10 3 8 10 10 8 18 13 5 7 3 16 5 9 5
levels of X 150/mm3. However, there was no quantitative correlation of blood and tis sue levels in individual subjects (r = 0.13, p > 0.2). There was even less of an individual correlation between blood eosinophil levels and the tissue eosinophil response to 48/80 (r = 0.07, p > 0.2). At the sites of injection of the lowest ragweed dose inducing a posi tive wheal reaction, eosinophil responses of more than minimal degree were seen in only 1 of 8 subjects tested in threshold dilution studies. The number of mast cells in the test sites appeared to vary inversely with the de gree of eosinophil response although there was significant variation in this regard. For this reason, the negative correlation be tween eosinophil and mast cell counts after ragweed injection (r = -0.33) was not sta tistically significant. It should be noted that
Table II. Correlations between tissue eosinophil re sponses and other parameters Primary measurement
Correlate measurement
Tissue eosinophil response antiragweed antibody to ragweed antiragweed Tissue eosinophil response antibody to 48/80 Tissue eosinophil response blood eosinophil to ragweed level Tissue eosinophil response blood eosinophil level to 48/80 Tissue eosinophil response tissue mast cell response to to ragweed ragweed
r value 0.63 0.46 0.13 0.07 -0.33
it was not feasible, at the level of resolution possible, to distinguish partial degranulation of mast cells. Therefore, all mast cells with any granules were recorded as being pre sent. The levels of IgE class antiragweed anti body measured by the RAST technique were performed in sera obtained at the time of intradermal injection and have been re corded in another study [4]. Comparison of these levels with the eosinophil responses to ragweed in individual subjects showed a sig nificant correlation (r = 0.63, p
Factors in the Tissue Eosinophil Responses in Human Immediate Hypersensitivity Reactions Burton Zweiman, Robert I. Slott and Paul C. Atkins Allergy and Immunology Section, VA Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract. Factors underlying the eosinophil responses in the tissue sites of immediate hypersensitivity reactions have been investigated. A quantitative assessment of the number of eosinophils appearing in skin test reactions to ragweed antigen and compound 48/80 in rag weed-sensitive humans has been compared with several other parameters. There was a moderate, statistically significant, correlation with serum levels of IgE-class antiragweed antibody; also, eosinophil responses were minimal or absent in minimally positive threshold dilution skin tests. Tissue eosinophil responses were generally limited to those with baseline blood eosinophil levels of at least 150 mm3; however, there was no correlation between blood and tissue eosinophil levels in individual subjects. These findings suggest that the pathogenesis of the observed tissue eosinophil responses may be multifactorial.
Received: February 12, 1976.
carried out for several years sequential his tologic studies of cellular inflammatory res ponses in immediate hypersensitivity reac tions in human skin [3, 4]. The cellular res ponses we have seen are qualitatively simi lar to those present in many clinical allergic reactions. Recently, we have utilized modi fications permitting a more quantitative as sessment of the eosinophil response. It has, therefore, been feasible to compare the de gree of such responses in antigen-induced reactions with the degree of allergic reactivi ty of the host, the blood eosinophil level at the initiation of the reaction, and cellular Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:04:55 PM
Although it has long been known that prominent eosinophilic infiltration is com mon in tissue sites of human allergic reac tions, the factors underlying the degree of such eosinophilic responses in an individual patient are not well defined. Several lines of investigation in recent years have uncovered several factors with in vivo chemotactic ac tivity for eosinophils [1] with at least one factor likely of mast cell origin [2], Compa rable in vivo kinetic studies of allergic res ponses in the human respiratory mucosa have been limited by ethical and logistic considerations. For these reasons, we have
49
Zweiman/Slott/Atkins
Materials and Methods Patient Group 15 atopic subjects, ages 21-30, exhibiting strong wheal and flare reactions 20 min after intradermal testing with 20 PNU of mixed ragweed extract (prepared in the Allergy and Immunology Clinic, University of Pennsylvania) were studied, at times other than the ragweed pollinating sea son. No subjects were receiving symptomatic med ication or hyposensitization therapy at the time of study. Skin Test As described previously by us [4], threshold di lution skin testing with ragweed extract was car ried out in the lateral aspect of the upper arm, us ing intradermal injection of 0.02-ml aliquots con taining, respectively, 20, 6, 2, 0, 0.6, 0.2, 0.06, 0.02, 0.006, and 0.002 PNU. At another site, 0.02 of a 35 mg/ml solution of compound 48/80 (Burroughs-Wellcome, Research Triangle Park, N.C.) was injected intradermally. All gross reactions were read at 20 min by an individual unaware of the clinical history of the subjects. A reaction was considered positive if the mean wheal diameter was greater than 8 mm and at least twice that of the negative control.
found in these earlier studies that eosinophil res ponses were prominent by this time lasting for at least several hours thereafter. Biopsies were fixed in neutral bufferred formalin, sectioned at 6 pm and stained with May-Grunewald-Giemsa. Sec tions were coded and then overlaid with a translu cent nylon grid composed of squares 0.5 X 0.5 mm, permitting systematic counting of the eosino phils and mast cells in ten such grid squares (2.5 mm2) of dermis starting at the dermal-epidermal junction were recorded. Repeat readings of the same tissue section by the same observer showed good consistency with this method [4]. Selected biopsies were read independently by a second ob server as well, with good agreement between the independent readings. Statistical Analysis All the data from the different types of mea surement were tabulated separately at the conclu sion of the studies, before the codes on the serum specimens and tissue biopsies were broken. Corre lations were then attempted where appropriate, using a regression coefficient program on a Mon roe calculator.
Results
The total blood eosinophil levels, and tissue eosinophil responses/2.5 mm2 to the intradermal injection of ragweed both in the dose of 20 PNU and the extinction dilution are shown in table I. Also shown are the tis Blood Studies Immediately before the intradermal injection sue eosinophil responses to 48/80, and the of ragweed or compound 48/80, venous blood was tissue mast cell levels after injection of rag obtained for (1) total eosinophil count (counted weed and 48/80. It is apparent that there is with the Uno pipette system, Becton Dickinson considerable variation within the study Co.), and (2) serum specimens, coded and then an group of the tissue eosinophil responses to alyzed for antiantigen E antibodies of the IgE class by the Pharmacia Company, Piscataway, both ragweed and 48/80. Yet this was not N.J. simply a function of the blood eosinophil levels at the time of intradermal injections. Skin Biopsies It is true that tissue eosinophil responses 2 h after the injection, 3 mm diameter pinch biopsies were obtained at selected injection sites of more than 10 cells/2.5 mm2 were seen at ragweed extract, and compound 48/80, by tech ragweed 20 PNU skin test sites for the most part in those subjects with blood eosinophil niques previously described by us [3], We had
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:04:55 PM
responses in nonallergic reactions induced by compound 48/80. This report describes such comparative studies.
Zweiman/Slott/Atkins
50
Subject Blood eosino phil levels R.B. J.L. J.M. R.M. F.M. M.M. J.P. K.R. C.B. J.B. E.G. J.K. H.N. R.S. E.T.
193 320 306 288 384 400 176 256 158 80 144 96 112 144 112
Tissue eosino phil re sponse to ragweed 36 42 52 54 84 10 135 102 20 6 110 9 0 0 8
Tissue eosino phil re sponse to 48/80
Tissue mast cells
135 34 12 104 29 12 57 0 15 10 85 4 0 0 18
12 28 9 4 10 10 3 16 13 20 6 22 16 8 20
after after ragweed 48/80 10 3 8 10 10 8 18 13 5 7 3 16 5 9 5
levels of X 150/mm3. However, there was no quantitative correlation of blood and tis sue levels in individual subjects (r = 0.13, p > 0.2). There was even less of an individual correlation between blood eosinophil levels and the tissue eosinophil response to 48/80 (r = 0.07, p > 0.2). At the sites of injection of the lowest ragweed dose inducing a posi tive wheal reaction, eosinophil responses of more than minimal degree were seen in only 1 of 8 subjects tested in threshold dilution studies. The number of mast cells in the test sites appeared to vary inversely with the de gree of eosinophil response although there was significant variation in this regard. For this reason, the negative correlation be tween eosinophil and mast cell counts after ragweed injection (r = -0.33) was not sta tistically significant. It should be noted that
Table II. Correlations between tissue eosinophil re sponses and other parameters Primary measurement
Correlate measurement
Tissue eosinophil response antiragweed antibody to ragweed antiragweed Tissue eosinophil response antibody to 48/80 Tissue eosinophil response blood eosinophil to ragweed level Tissue eosinophil response blood eosinophil level to 48/80 Tissue eosinophil response tissue mast cell response to to ragweed ragweed
r value 0.63 0.46 0.13 0.07 -0.33
it was not feasible, at the level of resolution possible, to distinguish partial degranulation of mast cells. Therefore, all mast cells with any granules were recorded as being pre sent. The levels of IgE class antiragweed anti body measured by the RAST technique were performed in sera obtained at the time of intradermal injection and have been re corded in another study [4]. Comparison of these levels with the eosinophil responses to ragweed in individual subjects showed a sig nificant correlation (r = 0.63, p
