1. Changes in number of basophils.

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1 Changes in basphil cunt 123 Basphil respnse t antigen and anti-ige. 1. Changes in number f basphils. Yshir Tanizaki and Ikur Kimura 1) Divisin f Medicine, Misasa Medical Branch, 1) Secnd Department f Medicine, Okayama University Medical Schl Abstract: Bld basphils in subjects with brnchial asthma migrate frm bld stream int lcal allergic reactin sites after inhalatin f antigen int airways. The phenmena can be bserved by changes in number f basphils in the peripheral bld. The peripheral basphil cunt is at a nrmal level in nn-attack stage f asthmatics as in healthy subjects. The basphil cunt increases in pre-attack stages. and decreases during attack stages. These findings suggest that number f basphils changes in clse relatin t asthma cycle. and that by bserving number f basphils in the peripheral bld. it is pssible t detect near future attacks. The decrease in number f basphils and histamine release by stimulatin with antigen and anti-lge can be bserved in vitr in whle bld and basphils separated by density gradient centrifugatin and by cunterflw centrifugatin elutriatin. The decreased number f basphils in the peripheral bld represents migratin f the cells int lcal allergic reactin sites. Key wrds: basphils. brnchial asthma. asthma cycle. histamine release Intrductin Asthma attacks are elicited by bridging f IgE receptrs n mast cell membrane within 30 minutes after inhalatin f antigen 1-3) After chemical mediatrs are released frm mast cells, and immediate allergic reactins are induced by these mediatrs, varius bld cells including basphils migrate int allergic reactin sites in the airways f asthmatics, accmpanied with r withut late asthmatic reactin. It is well knwn that basphils release histamine, ne f the main chemical mediatrs released frm mast cells, n expsure t antigen and anti-ige+- 7 ). Basphils, hwever, have t migrate frm bldstream int lcal allergic reactin sites t act as the target cells f IgE antibdies. Therefre, changes in number f basphils in the peripheral bld are bserved in relatin t migratin f the cells int lcal allergic reactin sites. In the present study, changes in number f basphils were discussed in relatin t IgE-mediated

2 124 Changes in basphil cunt allergic reactin. Observatin fr changes in number f basphils in viv 1) Staining slutin fr direct cunt f basphils Basphils can be easily cunted by a staining slutin fr direct cunt f basphils and esinphils 8, 9). The staining slutin cmprises 11 mp f 0.05% tluidine blue slutin (0.05 g tluidine blue (Merck) in 50 mp f 1.8% sdium chlride, 22 mp f 96% ethylalchhl and 28 mp distilled water), 0.8mP f 0.03% light green slutin (Merck), 0.5mP f saturated slutin f sapnin (sapnin white, Merck) and 5 mp f 1/15 M phsphate buffer (ph 6.4). The slutin is stable and basphils are well differentiated within ten hurs after stained with the slutin. After staining with the slutin, number f basphils is cunted in a Fuchs-Rsenthal chamber. 2) Increase in number f basphils in preattack atages Number f basphils In the peripheral bld increases in pre-attack stages. Observatin fr an increase in number f basphils makes it pssible t detect fllwing attacks, and it is suggested that the future attacks will appear in almst asthmatics when the basphil cunt is higher than the average upper limit (60-65/cmm)B-IO). A pint, i. e. 65/cmm is designated as the 'attackthreshld' in asthmatic subjects, and when the basphil numbers exceed it, the subjects may develp an asthma attack with very near future. Furthermre, the attack-threshld is quite cnstant in each individual. An increase in number f basphils befre asthma attacks is als bserved in patients with sea squirt asthma at pre-attack stsges f wrking perids 11, 12). In clinical curse f natural asthma attacks, an increased number f basphils is ften bserved during attack seasn (Fig. O. 100 Fig. 1. Fig. 2. BasPhil '. 0 Asthmatic ltack ~L.-I-'-"6~ '~12:------:1':'"8 Mnths Changes in number f basphils In a 13-year ld by receiving lng term therapy 3) Decrease in number f basphils in attack atages Basphils increasing in pre-attack stages shw a tendency t decrease when attacks cme ut. When attacks imprve and number f basphils still shw a lw level, the patient des nt develp near future attack. Number f basphils, hwever, Increases again after imprvement f attacks, the patient may develp near future attack (Fig. 2). 150 ~ 100 I l: CD Attack threshld Tim (htji.sl I I Attack: Attack in r-- stage --1., near furure V Attack lr.. Crrelatin between changes In number f basphils and asthma attacks (a shema).

Changes in basphil cunt 125 4) Migratin f basphils int lcal allergic reactin sites The decrease in number f basphils during attack stages shws migratin f the cells frm bldstream int lcal allergic

3 Changes in basphil cunt 125 4) Migratin f basphils int lcal allergic reactin sites The decrease in number f basphils during attack stages shws migratin f the cells frm bldstream int lcal allergic reactin sites. The basphil migratin is bserved at skin sites f intradermal allergen injectin by the applicatin f cantharides plaster 13) A slightly higher number (2.9%) f basphils is fund when exudate is derived frm an area f skin demnstrating a psitive reactin t an allergen. On the cntrary, analysis f vesicular fluid frm areas f untreated skin shws a lwer appearance f basphils (1.1%). These findings suggest that basphils tend t emerge in greater numbers at sites f intradermal allergic reactins than at ther skin sites. In healthy, nn-allergic subjects, fewer basphils (0.3%) are fund in the vesicular fluid ver areas f untreated skin. Basphil migratin int tissuse is als bserved in sputum during symptmatic episdes f brnchial asthma, particularly during the last phase f attacks. At the imprvement f attacks, when the patient is cmpletely asymptmatic, basphils are n lnger fund in the sputum I.). Observatin fr changes in number f basphils in vitr 1) Separatin f basphils frm peripheral (l) bld Separatin by density gradient centrifugatin Basphils can be highly separated by nly ne time centrifugatin with density gradient using buffy cat frm 6 me f whle bld, and nly 1 me venus bld is enugh t separate when the whle bld methd is used. The purity f basphils averaged 22.7% in buffy cat and 15% in whle bld 15). (2) Separatin by cunterflw centrifugatin elutriatin Basphils can be als separated by cunterflw centrifugatin elutriatin using a JE-6B rter (Beckman)16.m. The frequency f varius leuccytes cllected int each fractin by this methd was previusly describbed 18). The frequency f lymphcytes is higher at a lw flw rate (4.5 t 8 me/min), and gradually decreased as the flw rate increases. The frequency f neutrphils is lw at a lw flw rate, markedly increases at a flw rate f 10 mf/min and reachs a peak at a flw rate f 13 mf/min. The frequency f basphils and esinphils increases at a flw rate f 8 lomf/min, and 12 rnf/min r higher, respectively. The purity f basphils (0.9±0.5%) in the peripheral bld gradually increases at a lw flw rate and reachs a peak (l1.8±2.0%) at a lw flw rete f 9 me/min. The viability f basphils is greater than 95% in all fractins, and degranulatin is nt bserved in any basphils by the direct cunting methd 8-10). The cells well mved under phasecntrast micrscpic bservatin 19, 2(J). 2) Decrease in number f basphils in vitr Basphils reacting t allergen shw mrphlgical changes, pear-shaped (riented mvement) and swllen type (degranulatin), and the number f basphils decreases, accmpanied with histamine release 19) In ur previus studies, reactive basphils (increase in mtility) increased frm 11.7% befre incubatin with antigen t 45.8% after stimulatin with the antigen. At the same time, the number f basphils (39/cmm) decreased t a significantly lwer level (lo/cmm). The mean percent decrease was 74.5% and the mean histamine release frm basphils was 53.4%.

4 126 Changes in basphil cunt References 1. Cnry MC, Adkinsn NF, Lichtenstein LM. Measurement f IgE n human basphils: relatin t serum IgE and anti-igeinduced histamine release. J Immun : , Ishizaka T, Ishizaka K, Cnrad DH, Frese A. A new cncept f mechanisms f IgE-mediated histamine release. J Allergy Clin Immun1. 61 : 320, Ishizaka T. Analysis f triggering events In mast cells fr immunglbulin E mediated histamine release. J Allergy Clin Immunl. 67 : 90-96, Radermecker MF. Allergen-induced histamine release frm whle bld. Clinical evaluatin. Int Archs Allergy Appl Immun1. 63 : , Tanizaki Y, Kmage H, Mrinaga H, Kitani H, Gda Y, Kmura 1. Allergen- and anti-ige-induced histamine release frm whle bld. Int Archs Allergy Appl Immunl. 73: , Tnizaki Y, Sud M, Kitani H, Kawauchi K, Mifune T, Takahashi K, Kmura 1. Basphil reactivity t anti-ige and allergen in asthmatic subjects; relatin t huse dust allergy. Jpn J Clin Immun. 7 : , Tanizaki Y, Kmage H, Sud M, Mrinaga H, Kitani H, Nakagawa S, Takahashi K, Kimura 1. Reactivity f sensitized human basphils as expressed by histamine release. Jpn J Allergl. 33 : , Kimura I, Mritani Y, Tanizaki Y. Basphils in brnchial asthma with reference t reagin-type allergy. Clin Allergy 3 : , Tanizaki Y. Studies n basphilic leuccytes. 1. An imprved methd fr a simple direct methd cunting abslute basphils and esinphils, and its clinical significance. Okayama Iggakai Zasshi 85 : , Kimura I, Tanizaki Y. Changes f basphilic leuccytes f the peripheral bld in brnchial asthma, with reference t the threshld f asthmatic attacks. Jpn J Allergl. 19 : , Kimura I, Mritani M, Tanizaki Y, Sait K. Changes f basphils in reagin type allergy (sea squirt asthma). Jpn J Allergl. 20 : , Tanizaki Y. Studies n basphilic leuccytes. 2. Changes f basphilic leuccytes in brnchial asthma. Okayama Iggakai Zasshi 85: , Kimura I, Tanizaki Y, Takahashi K, Sait K, Ueda N, Sat S. Emergence f basphils at sites f lcal allergic reactins using a skin vesicle test. Clin Allergy 4: , Kimura I, Tanizaki Y, Sait K, Takahashi K, Ueda N, Sat S. Appearance f basphils in the sputum f patients with brnchial asthma. Clin Allergy 1 : 95-98, Tanizaki Y, Takahashi K, Hskawa M, Ishibashi K, On H, Gda Y, Nakamura Y, Sasaki Y, Kbayashi M, Kimura 1. Purificatin f basphilic leuccytes frm peripheral bld. Acta Haematl Jpn. 41 : Jeminek JF, Cntreras TJ, French JE, Shields LJ. Technique fr increased granulcyte recvery frm human whle bld by cunterflw centrifugatin elutriatin. 1. In vitr analysis. Transfusin 19 : , Glick D, Redlich DV, Juhs ET, Mcewen

Name: Date: Period: Notes: The Blood and Lymphatic System

Name: Date: Period: Notes: The Blood and Lymphatic System Name: Date: Perid: Cmpsitin f Bld and their Functins Red Bld Cells (aka ) Structure Ntes: The Bld and Lymphatic System D nt have a like ther cells d Cntain a specialized prtein called Hemglbin cntains