Int. Archs Allergy appl. Immun. 53: 78-87 (1977)

Inhibition of Reagin-Mediated PCA Reactions in Monkeys and Histamine Release from Human Leukocytes by Human IgG4 Subclass' H. M. Vijay and L. Pereimutter Drug Research Laboratories, Health Protection Branch, Health and Welfare Canada, Ottawa, Ont.

Abstract. Human myeloma proteins of IgG4 subclass in contrast to myeloma proteins IgGi, IgGa and IgG3 , were capable of blocking PCA reactions in monkeys mediated by human reaginic antibodies of IgE class. In addition to IgE, IgG.i myeloma protein was also capable of sensitizing leukocytes from normal individuals and gave histamine release (HR) upon challenge with anti-human IgG,i. Leukocytes from 11 allergic individuals and from 9 normal subjects sensitized with the serum of allergic patients, were capable of releasing histamine with anti­ human lgG.i, anti-human IgE, and the specific allergen. No response was obtained with anti­ human IgGi and IgG3 sera. Leukocytes from the normal individuals released histamine from 3 to 20% with anti-human IgG4 and from 6 to 30% with anti-human IgE. Moreover, normal leukocytes sensitized with lgG4 myeloma protein or a serum of an allergic patient heated at 56°C for 2 h. released a significant amount of histamine on challenge with anti-human IgG4 whereas no response was obtained with anti-human IgE. The biological role of human IgG4 in immediate hypersensitivity reactions is discussed in relation to human IgE.

Although a number of investigators have demonstrated that human reagins are asso­ ciated with IgE immunoglobulins [1-3], there have been recurring reports of reagin activity associated with other immunoglobu1 Presented in part at the annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. April, 1975 and at the Canadian Federation of Biological Societies, Winnipeg. Man: June. 1975. Received: December 15, 1975.

lins [4-7]. Recently, inhibition of reaginmediated PCA reactions in baboons by hu­ man IgG4 and in monkeys by human IgG2 has been reported by Stanworth and Smith [8] and Malley et al. [9], respectively. The present study was undertaken to de­ termine if reaginic activity is associated with any of the human IgG immunoglobulin sub­ classes. For this purpose, a study was per­ formed on the ability of antisera to human IgG,, IgGs and IgG4 to release histamine from leukocytes of allergic patients sensitive Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:20 AM

Introduction

to various allergens. The preliminary find­ ings have been reported earlier [10]. In ad­ dition, reagin-mediated competitive PCA in­ hibition experiments were performed in monkeys with human myeloma proteins of the IgG subclasses and IgE immunologlobulins. The results of this study indicate that in addition to human IgE, IgG4 was specifical­ ly capable of sensitizing human leukocytes and blocking the binding of human reaginic antibodies of IgE class to monkey mast cells. It was also shown that some of the re­ aginic activity was associated with IgG4 im­ munoglobulin. Materials and Methods Hitman Allergic Sera Sera from 11 patients allergic to grass, rag­ weed, trees, alternaria, and cat hair were em­ ployed in this study and obtained from Dr. H. Dehejia, Ottawa. Ont. The sera were stored in small aliquots at -70 C. Human Myeloma Proteins Myeloma proteins of each of the four human IgG subclasses were kindly supplied by Dr. B. J. Underdown, University of Toronto, Toronto. Ont. Total normal human IgG was purchased from Nordic Immunological Laboratories. Tilburg, Netherlands. Antisera Heavy chain specific antisera directed against human IgG subclasses were obtained commercial­ ly, i.e. swine anti-IgG,, anti-IgG,, and anti-IgG4 from Nordic Immunological Laboratories. Sheep anti-human IgG4 was obtained from Dr. B. J. Un­ derdown of Toronto. Goat antisera against human IgE and IgG were obtained from Hyland Labora­ tories, Costa Mesa, Calif. Gel diffusion analysis of anti-human lgG 4 with varying concentrations (0.1, 0.2, 0.4, 0.8, and 1 mg protein) of human IgG ,/, IgG,K. IgGsK, and IgE did not show any detectable precipitin

79

bands, whereas there was a strong band against human IgG4K. Anti-human IgE was also found devoid reactivity to human K- and /-chains. Quantitative precipitin analysis [11] revealed that anti-human IgE contained I mg and anti-human IgG, 0.89 mg antibody protein/ml. Antigens A water-soluble extract of short ragweed pol­ len (WSR) was prepared as previously reported 112] and a water-soluble extract of Kentucky blue grass (WSG) was kindly supplied by Dr. A. K. M. Ekramdullah, University of Manitoba, Winnipeg, Man. Water-soluble extracts of white birch (WSB; 1:20), alternaria (ALT; 1:10), and cat hair (CH; 1:10; w/v) were purchased from Hollister-Stier Laboratories, Missisauga, Ont. The extract of white birch (1:20) was extensively dialyzed against water and lypholized. The extracts of alternaria and cat hair were used in their commercial dilu­ tion form of 1:10 each. Histamine Release (HR) from Human Leuko­ cytes A modified technique of Lichtenstein and Os­ ier [13] was employed to determine the ability of antisera to various subclasses of human IgG to re­ lease histamine from leukocytes of individuals al­ lergic to various allergens as well as from normal volunteers. Leukocytes from 11 allergic patients were challenged with antisera to human IgG,, IgG3, IgG„ total IgG, and IgE and the specific allergen(s). In addition, leukocytes from 2 normal individuals were passively sensitized with sera of allergic patients (Hu and Mer) and challenged with antisera to the afore-mentioned immunoglob­ ulins and the specific allergen [14]. Leukocytes from normal individuals were also sensitized with 100//g of human IgG,, IgG3 and IgG, myeloma proteins and challenged with the antisera to hu­ man IgG,, IgG3, IgG,, total IgG and IgE. Allergic Serum Transfer Test in Monkeys The titer of reaginic antibodies in the sera of allergic patients was determined by passive cuta­ neous anaphylaxis (PCA) reactions on rhesus Ma­ caco mulatto (3-5 kg) [15], In each experiment. 0.1 ml of allergic serum was injected intradermalIy (i.d.) on the abdomen of a monkey at seven concentrations (1/10, 1/100. 1/250, 1/500, 1/1.000,

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Vijay/Perelmutter

80

Vijay/Perelmutter

Table I. HR from leukocytes of allergic individuals upon challenge with various allergens Allergic patients

Patients allergic to

Percent release of histamine*1from leukocytes of allergic individuals upon challenge with WSR 0.03-3 /tg/ml

Sah Lau Bar Ha Vog Hu Ric Pa Ell Bai Mcr

WSR, WSG WSR, WSG WSR, WSG WSR, WSG WSR, WSG WSG, WSB WSG, WSB WSR, WSG, WSB WSG ALT CH

WSG 0.03-3 /tg/ml

WSB ALT 0.03-3 /tg/ml 1-100 _

_

-

—

-

-

49 20 30 60 30 90 4 0

16 100 30 17 36 41 68 38 90 0

70 40 38 -

-

-

-

-

CH 1-100 _ —

-

-

-

-

48 3

-

-

33

- = Not determined. 1 The optimal concentrations of whole aqueous extracts of ragweed, grass, and birch white pollen (WSR, WSG, and WSB), respectively, were used. The preparations of alternaria (ALT), and cat hair (CH) were employ­ ed in different dilutions rather than concentrations.

Competitive Inhibition Experiments in Monkeys Myeloma protein of each of the four human IgG subclasses, total normal human IgG, and IgE were used in reagin-mediatcd competitive inhibi­ tion testing. Immunoglobulin solution (0.05 ml) was injected (i.d.) into the shaved abdomen of a normal monkey 4 h prior to injection (i.d.) of an equal volume of sensitizing serum into the same site. A reference reaginic serum, diluted up to 1:10, from a ragweed pollen-sensitive individual with a total serum IgE level of 1,250 U/ml and a 24-hour P-K activity of 1,000, was used for pas­ sive sensitization. After 18 h, the animal was given

an intravenous injection of 5 mg WSR (in 0.5 ml saline) and 4.5 ml 2% Evan’s blue dye. The mean diameter of blueing was recorded 30 min after the intravenous challenge. In another experiment, a mixture of equal volumes of immunoglobulin (0.05 ml) and the sensitizing serum (0.05 ml) was injected (i.d.). Reverse Passive Cutaneous Anaphylaxis in M on­ keys A monkey was injected (i.d.) with 0.1 ml (100,«g) of the myeloma protein solutions to be tested. After 4- and 27-hour period, the animal was injected intravenously with 0.25 ml of sheep anti-human IgG4 and 4.5 ml 2°/o Evan’s blue dye. Skin sites were examined 30 min after intravenous challenge. Radioimmunoassay A Phadebas IgE test kit (Pharmacia Lab, Mon­ treal, Que.) was used to determine whether IgG4 myeloma protein contained IgE protein. It was found that the IgG4 myeloma protein at a concen­ tration of 2 mg protein was negative for IgE at a level of 100 ng.

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1/2,000, and 1/4,000). After 24 h, the animal was given an intravenous injection of 5 mg antigen (in 0.5 ml saline) and 4.5 ml 2°/o Evan’s blue dye. The reactions were recorded 30 min after the intrave­ nous challenge. The titer of each serum was ex­ pressed as the reciprocal of the highest dilution giving the diameter of the blueing reaction greater than 5 mm. The monkeys were recycled at interval of 3 months and consistent responses were ob­ tained with the standard serum (Sah) in each ex­ periment.

81

Inhibition of Reagin-Mediated PCA Reactions and Histamine Release

Results It is evident from table I that histamine was specifically released from leukocytes of allergic individuals on challenge with the specific allergens. Table II. HR from leukocytes of allergic individuals upon challenge with anti-human IgE, IgG, and IgG.i Allergic patients

Percent release of histamine from human leukocytes upon challenge with anti-IgE anti-IgG anti-IgG.i 1:10,000-1:100 1:100 1:10,000-1:100

Sah Lau Bar Ha Vog Hu Ric Pa Ell Bai Mer

7 28 7

41 100 30 40 20 25 50 50 61 21 30

19 53 19 4 15 70 49 27 61 31 60

-

-

8 -

15

_ = Not determined.

Table II shows the results of HR from the above patients on challenge with antis­ era to human IgE, IgG, and IgG4. Signifi­ cant amounts of histamine were released from the leukocytes on challenge with anti­ human IgG4 and anti-human IgE, while lower responses were obtained with total anti-human IgG. In some cases, such as leu­ kocytes of patients Hu, Bai, and Mer, who are allergic to trees, mold, and cat hair, re­ spectively, a greater amount of histamine was released on challenge with anti-human IgG4 than with anti-human IgE. In others, anti-human IgE released an amount of his­ tamine equal to or greater than anti-human IgG4. Further, a high concentration of whole anti-human IgG caused HR up to 28%. No histamine was released (