Dietary nucleoside-nucleotide mixture aggravates nasal allergic responses induced by toluene diisocyanate in mice

NutritionResearch, Vol. 17. No. 10, pp. 1597~1605.1997 Copyright8 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0271~5317m %17.00+.m

PII SO271-5317(97)00154-l

ELSEVIER

DIETARY NUCLEOSIDE-NUCLEOTIDE MIXTURE AGGRAVATES NASAL ALLERGIC RESPONSES INDUCED BY TOLUENE DIISOCYANATE IN MICE Clement K. Ameho. MSc. Andrew A. Adjei, MSc , Keiko Yamauchi. MSc. Liu Asato. MSc., Shige Kakinohanal ‘, RN., Anil D. Kulkami’. PhD.. Y uzo Hire?, PhD. and Shigeru Yamamoto. PhD* Department of Nutrition, Department of Nursing, University of the Ryukyus, Nishihara-cho, Okinawa 903-01, Japan, Department of Surgery, St. Louis Medical Center, St. Louis. U.S.A., and Department of Nutrition and Biochemistry. Nakamura Gakuen College, Fukuoka. Japan. ABSTRACT In this study we evaluated the effect of a dietary nucleoside-nucleotide mixture (NNM) on nasal allergy induced by toluene diisocyanate(TD1) in mice. Female ddY mice were randomized into two groups and fed a nucleic-acid free 20% casein (control) or this diet suppIemented with NNM for 4 weeks. On the 4th week, mice were divided into 2 sub-groups; NNM sensitized, NNM- nonsensitized, controlsensitized and control-nonsensitized. The sensitized groups of mice were treated with two courses of intranasal application of 5% TDI in diethyl acetate for 5 consecutive days each time, separated by one week rest. Nonsensitized group of mice were similarly treated with vehicle. A week after the second sensitization all the treatment groups were provoked by applying 2.5% of TDI in vehicle. Nasal responses of sneezing, rhinorrhea, itching and snort at provocation, were scored for ten minutes. Degree of hair loss from the snout of mice was also scored for the length of the experiment. Lung and serum IgE were measured by ELISA. Results show that the sensitized groups of mice from each dietary group sneezed more frequently than the nonsensitized groups (p< 0.05). The NNM sensitized group sneezed more frequently that was different (p< 0.05) compared to the rest of the groups. Nasal responses for the sensitized groups were higher relative to the nonsensitized groups, with the NNM sensitized group having an overall higher nasal response than the rest of the groups. Both sensitized group of mice showed a higher concentration of lung and serum IgE compared to the non-sensitized groups (~0.05, with the NNM sensitized group having an overall higher IgE concentrations in both tissues compared to the rest of the groups (pc 0.05). We conclude that dietary NNM may aggravate nasal allergic responses in the predisposed. Key words: Nasal allergy, Nucleoside-nucleotide Nasal response.

mixture, Toluene diisocyanate.

INTRODUCTION Much interest has been generated in recent years towards understanding the pathophysiology of hyperimmune (allergic) reactions. In different individuals allergy may take different forms such as hayfever, urticaria (hives), chronic eczema (skin irritation), bronchial asthma, hypersensitive pneumonitis and nasal allergy, which are characterized by hyper-responsivness of the airways ( 1, 2). Nasal allergy is characterized by sneezing, nasal blockage and nasal discharge. * To whom Correspondence

should be addressed. 1597

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C.K. AMEHO et al.

Various mediators like leukotrienes (LT), histamine, prostaglandin (PGs), platelet-activating factor (PAF), cytokines, kinins-bradykinins and N-alpha-tosyl-L-arginine (3) have been implicated in the development of symptoms. Histamine, the well known allergic mediator causes constriction of intestinal and bronchial smooth muscles, and may contribute to increased peristalsis of bronchospasm associated with food allergies and asthma respectively. However in these instances, especially in asthma, antihistamines are not effective in blocking the reaction (1). The role of LT antagonists in the treatment of immediate hypersensitivity has also shown limited clinical usefulness(4). It remains important therefore for an effective alternative prophylaxis to be found for the control and/or treatmemt of hypersensitive reactions. Most implicated mediators in hypersensitive reactions are also involved in both cell mediated and humoral immunity. The relationship of nucleic acids and/or their components in immune functions is becoming increasingly evident. Dietary nucleotides and nucleosides have been shown to enhance in vivo and in vitro cell mediated immunity, augment the immune response to cardiac allograft survival (5,6), enhance lymphocyte proliferation, and interleukin-2 production (7) and improve the response to sepsis in mice (8). Dietary nucleotides and nucleosides also increases the delayed hypersensitivity to chemical and bacterial antigens and xenoantigens (9, 10, 11). Typically, nucleoside-nucleotide mixture stimulate the production of T-cells which secrete a number of the mediators involved in hypersensitive reactions, as in nasal allergy. The effects on the humoral immune system has also been well documented. It has been shown that dietary nucleotides and nucleosides increased antibody production, gamma interferon and B-cell number in both humans and mice ( 12, 13). Conversely, nucleotide-free diets (NFD) have been found to have the opposite effects on the same indeces. On the basis of these results and others, one can reasonably speculate that nucleic acids and their components have stimulating effects on the immune system. Thus it seems plausible that controlling the dietary intake of nucleic acids which serves as a precursor for the synthesis of these immune factors presents a possibility for the control of hypersensitive reactions in the predisposed. Recent studies have documented that the body depend mainly on the salvage of purine and pyrimidine bases and that the de novo synthesis is not adequate to meet the demands of various metabolically active cells or tissues ( 14). However, most food substances contain appreciable levels of nucleic acids in the form of nucleoproteins and these may be adequate to enhance the production of implicated immune factors that potentiate hypersensitive immune reactions. Thus we postulated that dietary limitation of nucleic acids and/or their precursors, or total deprivation during periods of heightened hypersensivity may be of value in the treatment of allergic reactions. Accordingly, we examined the effect of nucleoside-nucleotide mixture diets on nasal allergic reactions. We utilized this murine model of allergic rhinitis because it has been used extensively to assess known human respiratory sensitizers (15)

MATERIALS

AND METHODS

Animuls und diets Specific pathogen free female ddY mice weighing 3 l.O-34.3g obtained from Kyudo breeding laboratories (Kumamoto, Japan) were used for the experiment. Four-week old mice were kept in a constant temperature (25 & 2°C) and humidity (50-70%) with a 12hr light period. Animal care was in complaince with applicable guidelines from the Ryukyus University policy on Animal Care and Use. The mice were allowed to adapt to our laboratory environment for one week before the onset of the experiment, during which period they were maintained on a commercial non-purified diet (Nihon clear, Osaka, Japan). This standard mouse diet contains 25.5% pr&ein and 43% fat by weight. After one-week weight-matched mice were randomly assigned to two dietary groups; 20% casein (control) or 20% casein supplemented with 0.5% nncleoside-nucleotida mixture (NNMgroup) for four weeks. NNM is a mixture of nucleosides and a nucleotide consisiting of inosine (8g/L), guanosine monophosphate (12.2g/L), cytidine (7.3g/L), uridine (S.Sg/L) and thymidine ( l.@/L) at a molar ratio of 4443: 1 respectively. The diets were made isonitrogenous and isocaoric by adding appropriate amount of glycine. The composition of the diets are shown in table 1. 0x1 the

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MIXTURE AND ALLERGY

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4th week, mice from each dietary group were again divided into two sub-groups to form a total of four treatment groups: control-sensitized, control-nonsensitized, nucleoside-nucleotide mixture sensitized, and nucleoside-nucleotide mixture nonsensitized, depending on whether they were treated with toluene diisocyanate (TDI) in diethyl acetate (sensitization) or with the vehicle diethyl acetate (non-sensitization). Previous studies had shown that 0.5% of NNM fed for 4 weeks results in maxumum response. Sensiticatinn and Provocution. Sensitization was done by the method of Tanaka et al. (16) with slight modification (Fig 1). Mice were sensitized by dropping 2ul of 5% TDI (Wako, Japan) dissolved in ethyl acetate from an autopipette into the nostrils of mice under slight ether anesthesia for five consecutive days. This was repeated following a week of rest. Non-sensitized mice were similarly treated with vehicle. Mice were again allowed one week rest after which all groups were provoked with 4ul of 2.5% TDI without anesthesia to induce nasal allergy-like symptoms. The nasal responses of sneezing, itching, watery rhinorrhea and snorting were scored for ten minutes by the method of Irifune et al. ( 17 ) (Table 2). Hair loss was scored by observation of the extent of hair loss from the snout of mice over the period of sensitization. All scorers were blinded to the treatment groups.

TABLE 1

Composition

Diets (%)

NNM

Material

Control

Casein

20.0

20.0

Q! -starch

44.4

44.3

Sucrose

22.2

22.2

5.0

5.0

Mineral mixture* Vitamin mixture**

1.0

1.0

Cellulose

2.0

2.0

Oil

5.0

5.0

Glycine

0.4

NNM*** *

of Experimental

0.5

Obtained from Oriental Yeast co. Tokyo. The composition was as follol\s: (mgikg) CaHPO, 2H,O, 7,289; KH,PO,. 12,800; NaH,PO,. 4680; Nacl, 2.330; Ca.lactate. 17.550: Fe&rate, 1,590;MgSO,, 3,590; ZnC0,,55; MnSO, 6Hz0. 60: CuSO, 5H,O, 15: KI, 5; ** Obtained from Oriental Yeast company. The composition r\as as folio\\ s: (mpikg) Thiamine HCL, 12: Riboflavin, 40; Pyridoxine HCL. 8: Vitamin-B13.50, Ascorbic acid, 300: D-biotin, 0.2: Folic acid, 2; Calcium pantothenate, 5; p -aminobenzoic acid, 50; Niacin, 60; Inositol, 60; Choline chloride, 2ooO;dl- LYtocopherol acetate, 50; Menedione, 52: Retin! I acetate, 5WO and Ergocalciferol. looo. *** Nucleoside-nucieotide mixture; The composition was as follows: (g/L) Inosine, 230; Cltidine, 210; IGMP 7,Na,350; Uridine. 160; Thymidine, 50.

C.K. AMEHO et al.

1600

2nd sensitization

1st

Feeding diets 0

sensitization

Rest

I

I

I

4

5

6

Rest I 7

Provocation

Sacrifice 1 9

8

Weeks

FIG. 1. Protocol for TDI treatment. Mice were fed diets for 4 weeks, and sensitized with 5% TDI in diethyl acetate for 5 days, allowed one week rest , and resensitized again during the 6th week. Mice were again rested for a week after which they were provoked with 2.5% TDI. One week after provocation mice were sacrificed. Control mice were treated with diethyl acetate but provokedwith TDI.

Lung and serum IgE measurements One week after provocation, mice were killed by intraperitoneal administration of 10% nembutal (O.lml/kg body weight). Serum and lung were removed and immediately stored frozen for later analysis. IgE was quantified by mouse ELISA IgE kits (Yamasa Shoyu CO., Chiba, Japan) following the manufacturer’s instructions. Statistical arui~ysis Data was analysed by analysis of variance and Wilcox U-test. Duncans multiple range test was employed to determine significant differences among means at ~0.05. TABLE 2

Criteria for Scoring the Nasal Response of Mice

Nasal Response

SCORE 0

1

2

3

Itching

-

Mild

Rhonorrhea

-

At the nostril

Snort

-

Mild

Moderate

Severe

Hair Loss

-

Mild

Moderate

Severe

Moderate Between I & 3

Severe Drops of discharge from the nose

The scores were measured for ten minutes, except for hair loss which was scored over the period of the experiment.

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RESULTS Snee:ing reflex The results of nasal allergic rhinitis symptoms are shown in (Fig 2). Generally, the sensitized group of mice belonging to both NNM and control dietary groups sneezed more frequently at provocation than their corresponding nonsensitized groups. However, the NNM-sensitized groups sneezed more frequently that was higher (p
Sensitized

Nonsensitized

Sensitized

Nonsensitized

FIG. 2 Sneezing frequency of mice (n=12) in each dietary group. *vs the rest of the groups, pdO.05. **vs Control-Nonsensitized, p
TABLE 3 Scoring of Nasal Symtoms Induced by TDI in Mice Dietary groups

Nasal responses

NNM-sensitized

2.75 f 0.16*

NNM-nonsensitized

I so f 0.19**

Control-sensitized

1.63 + 0.27**

Control-nonsensitized

0.75 + 0.18

Values represent mean f SEM; n=12 in each dietary group, *vs the rest of the p
C.K. AMEHO et al.

1602 Nasal responses to provocation

The behavior score at provocation, measured as a total of the nasal responses of itching, watery rhinorrhea and snort, and degree of hair loss during the entire length of the experiment is shown in Table 3. Sensitized mice in each dietary group showed severer irritability at provocation (behavior score) compared to nonsensitized groups. The NNM-sensitized group showed a higher nasal response than the control-sensitized group. Similarly, the NNM-nonsensitized group showed higher nasal response compared to the control-nonsensitized groups. Overall, the NNM-sensitized groups showed greater nasal response compared to the rest of the groups.

a loo-

*

Sensitbxi

Nonsensitized

Sensitized

Nonse.nsitizd

b

FIG. 3. Lung (a) and serum (b) IgE concentrations of mice (n=12) in each dietary p&OS. group. * vs the rest of the groups, p4.05, ** vs Control-nonsensitized,

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Lung and serum IgE Figures 3 (a and b) shows results of lung and serum concentrations of IgE. In both tissues, both sensitized dietary group of mice had higher IgE concentrations compared to the nonsensitized dietary group of mice (~0.05). Overall, the NNM-sensitized group of mice showed a higher concentration of IgE in both tissues compared to the rest of the groups. Serum IgE concentrations were comparatively higher than Lung IgE concentrations in all the dietary groups of mice.

DISCUSSION The results of our study suggest that the presence of nucleosides and nucleotides in the diet may aid in the pathogenesis of allergy in the predisposed and/or aggravate symptoms in hyper-reactive individuals. Supplementation of diet with NNM mixture resulted in a significant elevation of all the outwardly observable markers of allergy in this murine allergic rhinitis model when compared to mice fed the NNM-free (control) diets (Fig 2 & Table 3). Sensitized group of mice showed more intense nasal allergic responses at provocation compared to the non-sensitized groups. Among the dietary groups, however, the different sneezing frequency and the high nasal response of the NNMsensitized group compared to the control-sensitized group on one hand, and the high nasal response of the NNM-nonsensitized group compared to the control-nonsensitized group on the other hand shows the differential effect of the NNM diet in increasing the degree of severity of allergic rhinitis in our model. The prolonged sneezing experienced by the NNM-sensitized group even after the designated time of measurement (10 minutes) also gives an indication of the effect of the diet. The two diets used in our study were isonitrogenous and isocaloric (Tablel) and differed only in their nitrogen presentation. The different forms of nitrogen gave clear differences in the response of the mice to antigen stimulation in our experiment and thus allowed us to evaluate the effect of these diets on allergic pathogenesis. The higher systemic concentrations of IgE was possibly released from the site of local production. The relatively high concentration of both serum and lung IgE observed in the non-sensitized groups in this study compared to other reported studies may be the result of either strain differences, age of mice and the mode of sensitization employed. Nasal allergy is considered to be an example of type 1 allergy, the pathophysiology of which is poorly understood. However, many studies have reported a host of mediators in immediate allergic responses. Notable among these are histamine, IgE and the products of the arachidonate cascade: LTC, LTD, and LTE,, PGD, and PAF. It has been shown that there are no preformed stores of arachidonic acid ( 18). Howecer, nucleotides apparently stimulate lipase activity (19) and causes the release of arachidonic acid from endogenous phospholipids, and subsequent synthesis of arachidonic acid derivatives implicated in nasal allergic response in whole tissues (20). NNM is alleged to have a putative role as a precursor for the synthesis of components of cell-mediated immunity (5). which secretes some of the implicated allergic mediators. and has also been shown to be involved in humoral immunity (13). Various other studies have reported that TDI sensitization enhances the synthesis of nervous pathway transmitters such as substance P and calcitonin gene-related peptide, and that neurogenic inflammation increases the turnover rate of histamine in nasal mucosa and lung (21, 22, 23). Interestingly, nucleotides have been implicated in central nervous system function (24, 25). Thus it is possible that NNM may have also enhanced the production of these mediators that resulted in the intense response shown by sensitized mice in that group. It is also possible that other mediators not directly related to NNM may have acted in concert to augment response in the mice. Presently, these are not determinable from our experiment. More research is needed to understand the mechanism of aggravation of nasal allergy responses by nucleotide-nucleoside mixtures. To our knowledge, this is the first study that has assessed the effects of nucleosidenucleotide mixtures on experimental allergic rhinitis in mice. Even though we have employed TDI sensitization in inducing nasal allergy-like symptoms, this is not different from the etiology of nasal allergy in humans. Sneezing and rhinorrhea which were elicited in the test animals were similar to clinical symptoms in human nasal allergy. From the results of our study, we conclude that NNM may contribute to the pathogenesis or severity of nasal allergy.

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Accepted

for

publication

August

13,

1997