Clin. exp. Immunol. (1986) 67, 611-616

The role of basophils and mast cells in cutaneousbasophil hypersensitivity reaction

T. KISHIMOTO, R. SODA, K. TAKAHASHI & I. KIMURA Department of Medicine,Okayama University Medical School, Okayama, Japan

(Acceptedfor publication 13 August 1986)

SUMMARY

Basophils are the main cell component in cutaneous basophil hypersensitivity (CBH)reactions, but the role of basophils and the factors which gather them into CBH reactionsites are unknown. To investigate these problems, we induced CBH reactions in guineapigs and observed basophils and mast cells in the skin reaction sites using light andelectron microscopy. Basophils infiltrated into CBH reaction site appeared at 5 h after thechallenge with antigen, increased till 48 h and decreased thereafter. On the other hand, thenumber of mast cells and their granules decreased after the challenge with antigen andreached a minimum at 48 h, but recovered at 96 h. The changes in the number of basophilsand mast cells were complementary. This result suggested that the granules of mast cellsmay have a factor to gather basophils into the CBH skin reaction site. Furthermore,basophils infiltrated in the CBH reaction site were degranulated by the rechallenge withantigen, which was considered to be by an anaphylactic reaction.

Keywords cutaneous basophil hypersensitivity basophil mast cell

INTRODUCTION

Cutaneous basophil hypersensitivity (CBH) reaction is a delayed hypersensitivity reaction that isdifferent from the classical type in that basophils seem to be dominant in the reaction site and thereaction itself is transient and without induration (Richerson, Dvorak & Leskowitz, 1967; 1970;Dvorak et al., 1970). The role of basophils in the pathogenesis ofCBH is not well understood. Togain insight into the role of basophils and mast cells in this reaction, we induced experimentallyCBH reactions in guinea pigs and studied the pattern of infiltration by basophils and mast cells.Infiltration of basophils into the site of CBH reached a maximum 24-48 h after the challenge andgradually decreased thereafter. On the other hand, mast cells and their granules decreased innumber during this period and recovered after 96 h. Degranulation of infiltrating basophils,induced especially with the rechallenge of the antigen, was observed by light and electronmicroscopy.

MATERIALS AND METHODS

Immunization and elicitation of CBH. Thirty female Hartley guinea pigs, 200 to 250 g, wereprimed for CBH by immunization in all four foot pads with 10 pg ovalbumin (OA) incompleteFreunds adjuvant (total volume 1 ml per animal). CBH skin reactions were elicited 7 days later on

Correspondence: Dr Takumi Kishimoto, Second Department of Internal Medicine, Okayama UniversityMedical School, 2-5-1 Shikatacho, Okayama, 700 Japan.

6ii

deplated abdominal skin by intradermal injection of 50 yug OA in 0-1 ml saline. Reacted skins wereobserved at 20 min, and 5, 12, 24, 48, and 96 h after the challenge with antigen. After measuring thediameter of erythema and induration, local reactive skins were extracted. At the same time, bloodwas obtained by cardiac puncture, and absolute basophil, eosinophil and total white blood cellcounts were done by using Kimura's specific staining method (Kimura & Tanizaki, 1969) by Fuchs-Rosenthal.

Rechallenge ofthe antigen to the CBH reaction site. At 24 h after the challenge with antigen, someof the guinea pigs were rechallenged with 50, 5, 0 5, 0 05, and 0-005 ,ug OA in 0-1 ml saline. Normalsaline (0-1 ml) without OA was used as a control. Rechallenged skins were observed for 20 min andwere then excised for light and electron microscopy. Degranulation of basophils and their extrudedgranules and vacuoles were observed in the 40 different fields by a light microscope ( x 1000).

Histology. Animals were killed with ether and the skin from the reaction site was excised andplaced in 255% glutaraldehyde in 0 1 M sodium cacodylate buffer, pH 7 2. Tissues were trimmed to1-5 mm size and further fixed in similar fixative for 2 h and then post-fixed with similarly buffered1% OS04 for 2 h. Specimens were dehydrated in graded alcohol and embedded in Epon 812. Semi-thin sections of about 1 gum were obtained and stained with 0 1% toluidine blue in phosphatebuffered-saline, pH 6 5, and with Giemsa in 2% sodium borate, and observed with a lightmicroscope ( x 1000). By toluidine blue and Giemsa staining, granules of basophils appear darkblue while those of mast cells are stained reddish purple. The difference between these two cells canthen be easily perceived by their staining pattern as well as the size of the granules. Eosinophils arestained light green.

Thin sections of grey-silver to silver range were also obtained and stained with uranyl acetateand lead citrate for electron microscopic examination.

RESULTS

Skin rash ofCBH. The CBH reactive skin developed erythema at 12 h after the challenge and thesize of the reaction reached a maximum of 24 h. It disappeared completely at 96 h. Induration didnot appear during this period.

Cellular composition ofblood. The number ofwhite blood cells, determined in blood obtained bycardiac puncture, ranged between 3500 and 4500 per Ml with normal differential. The number ofbasophils increased significantly between 24 and 48 h. The number of eosinophils had no significantchanges (Table 1).

Cellular composition of the reaction site. As in normal skin, fibroblasts constituted the largestfraction in the skin of the reaction site. This component showed no change during the course ofCBH (mean of 878+30 fibroblasts per 1000 high power fields (HPF)). On the other hand,additional infiltrating cells increased after challenge reaching a maximum of 846 per 1000 HPF at 24

Table 1. Leucocyte counts (WBC) in CBH reaction

Time after WBC Basophils Eosinophilschallenge (x 102/mm3) (x 102/mm3) (x 102/mm3)

20 min 39-9+6-0 14-8+1 5 91-8+795h 39-1+7-6 63+19 108-3+12212h 374+11 1 8-2+1 5 1223+20-924h 42-1+150 16-0+5-1 80-0+16-848 h 47-7+4-7 32-0+3-9 119-3+6-896 h 47.5 +11-6 11 0+12 117-3 +16-5

Number represents mean + s.d. for five experiments.Three guinea pigs were used in each experiment.

T. Kishimoto et al.612

The role ofmast cells in CBH reaction 613u,

-a 3000U')as0.I 200-c-en*

0 0-Time courseofter challenge 020'min 5h- 12 h 24h 48h 96h

0

o~~ H.0

0

)-oEz 0

Fig. 1. Cellular composition of skin in the course ofCBH reaction. The number of basophils (M) increased 5 hafter the challenge and reached a peak at 24 h, then gradually decreased. By contrast, mast cells (0) decreased for48 h, but recovered at 96 h.

h, and gradually decreasing to 438 per 1000 HPF at 96 h. This pattern was also noted for suchinfiltrating cells as mononuclear cells, neutrophils and basophils. Eosinophils, however, did notbehave like other infiltrating cells. Basophils appeared in the reactive skin after 5 h (18 per 1000HPF), reached a maximum (312 per 1000 HPF) at 24 h and decreased thereafter (Fig. 1). On theother hand, mast cells decreased gradually from the initial 38 per I1000 HPF to eight per I1000 HPF at48 h (Fig. 1). Most mast cells observed at 48 h after challenge showed scanty granule pattern. Thenumber of mast cells recovered at 96 h to 43 per 1000 HPF. Their granular pattern was normal bylight and electron microscopy.

0 0 0

C-)~~~~~~~~~~~~~~~~~-

Fig. 1. Celulrpomposiioahnge ofsinfinltheincouseofCBH ractionThehlenumerVrofsbansophils(o)inceaednigeaftro0the 50H)wsuechallengeadrahdapkat2hthen grdulldecnreainsied Byconfteraste mastiaceallsn(0) dfereasedmfor

sinfiltatin eise asd mononulear celnurohlfnbasophils.wronen4ifrn Eoinophilsylihtmcowever, di not00bhP ave likeotercinfitratingf egnlls. Basophilsappare inctheareactivhte deskinyafte 5nhi(8 pser 1000eHPF)ng,reac thed nmaximu (31bsphdecre10aP)s t2ednddcese.hrate Fg 1.O h

lihtanelcro0icocoy

100 ~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 0~0

~~~~ 200~~~~~~~~~~~~

0.~~~~~~~~~~~~~~~~~~~~~~~~~E

0 .0 00 .5 5 5100~ ~ ~ Ds o ehlegd nie ~g

0i.2 opooia hne fiflrtn aohl fe h ehleg.Vrosdniiso h nie

rechallenge, but tDoseuofereohablengedlantigenaseg)

614 T. Kishimoto et al.

Fig. 3. A basophil in the rechallenged CBH reaction site. Twenty minutes after the rechallenge, skin was excisedand studied by light microscopy. At this time, basophils had few granules and many vacuoles indicatingdegranulation ( x 5200).

Fig. 4. A basophil in the rechallenged CBH reaction site. The experimental design is similar to that of Fig. 3 onlytransmission electron microscopy was used. The granular membrane of this basophil fused and disappeared.This is the first step towards degranulation ( x 26000)

The role ofmast cells in CBH reaction

Fig. 5. A basophil in the rechallenged CBH reaction site. The experimental design is same as that of Fig. 4. Thisbasophil shows clear open secretion of granules ( x 25000).

Physiological role of infiltrating basophils. When the sensitizing antigen was rechallenged at theskin reactive site after 24 h, the erythema increased after 20 mmn of rechallenge. The number ofbasophils and their component of granules decreased. This decrease was the function of antigendose used for rechallenging (Fig. 2). The number of vacuoles in basophils also increased inproportion to the dose of antigen (Fig. 2). Figure 3 shows degranulated basophils by lightmicroscopy. By transmission electron miroscopy, degranulated basophils contained fewer granulesthat were in homogeneous in the density of their granules (Fig. 4). Figure 5 shows the release ofgranules from a basophil into the surrounding tissue.

DISCUSSION

The data presented here indicate that in the course of experimentally-induced CBH reactions,basophils infiltrate into the skin reaction sites. Concordant with this is an increase in bloodbasophils, reaching a maximum after 24-48 h. Conversely mast cells and their granules graduallydecrease after the challenge with antigen reaching a minimum at 48 h when the number of basophilsis maximum. Mast cells and their granules recover at 96 h as the number of basophils decrease.Several chemotactic factors could possibly be responsible for attraction to the skin site in CBH.Lymphocytes, such as T cells (Stadecker & Leskowitz, 1974) and B cells (Haynes & Askenase, 1977),as well as immune serum antibody (Askenase, 1973; Askenase et al., 1975; 1976) have beendescribed. In the case of lymphocyte-mediated CBH (Boetcher & Leonard, 1973; Ward et al., 1975),lymphokine is considered a factor attracting basophils, but no confirmation has been obtained. Inthe serum-mediated CBH, Haynes, Rosenstein & Askenase (1978) suggested that the sensitizingIgG of the guinea pig (IgGi1) on the surface of mast cells may cause degranulation ofmast cells, anda factor released during this process may serve as a chemotactic agent for basophils. The datapresented here are in line with this possibility, since basophilia intensifies as mast cells degranulate

6I5

6i6 T. Kishimoto et al.at the reaction sites. Kimura & Takaya (1979) have also shown cytoplasmic continuity betweenbasophils and mast cells in CBH reaction by transmission electron microscopy, suggesting that mastcells may serve as an attracting force for basophils. Accordingly we propose that mast cells'degranulation may have served in these studies to attract basophils to the reaction site. Theexperiments of basophil chemotaxis to the granules of mast cell are currently underway in ourlaboratory.

To study further the physiological action of basophils infiltrating into the CBH skin lesion,rechallenges with the antigen were done at the skin site 24 h after the initial challenge. The resultsindicated the morphological degranulation of basophils and this was an immediate reaction.Askenase et al. (1978) also demonstrated a similar phenomenon by using keyhole limpethaemocyanin sensitized CBH. We concluded that basophils infiltrated into CBH reaction site maybe sensitized.

REFERENCES

ASKENASE, P.W. (1973) Cutaneous basophil hyper-sensitivity in contact sensitized guinea pigs. I.Transfer with immune serum. J. exp. Med. 138,1144.

AsKENASE, P.W., HAYNES, J.D., TAUBEN, D. & DEBER-NARDO, R. (1975) Specific basophil hypersensitivityinduced by skin testing and using immune serum.Nature 256,.52.

ASKENASE, P.W., HAYNES, J.D. & HAYDEN, B.J. (1976)Antibody-mediated basophil accumulations incutaneous hypersensitivity reactions of guinea pig.J. Immunol. 117, 216.

ASKENASE, P.W., DEBERNARDO, R., TAUBEN, D. &KASHGARIAN, M. (1978) Cutaneous basophil ana-phylaxis: immediate vasopermeability increasesand anaphylactic degranulation of basophils atdelayed hypersensitivity reactions challenge withadditional antigen. Immunology 35, 741.

BOETCHER, P.A. & LEONARD, E.J. (1973) Basophilchemotaxis, augmentation by a factor from stimu-lated lymphocyte cultures. Immunol. Commun. 2,421.

DVORAK, H.F., DVORAK, A.M., SIMPSON, B.A.,RICHERSON, H.B., LESKOWITZ, S. & KARNOVSKY,M.J. (1970) Cutaneous basophil hypersensitivity.II. A light and electron microscopic description. J.exp. Med. 132, 558.

HAYNES, J.D. & ASKENASE, P.W. (1977) Cutaneousbasophil responses in neonatal guinea pigs: activeimmunization hapten specific transfer with smallamount of serum and preferential elicitation with

phytohemagglutinin skin testing. J. Immunol. 118,1063.

HAYNES, J.D., ROSENSTEIN, R.W. & ASKENASE, P.W.(1978) A newly described activity of guinea pigIgG1 antibodies: transfer of cutaneous basophilreactions. J. Immunol. 120, 886.

KIMURA, I. & TANIZAKI, Y. (1969) The stainingmethod and clinical evaluation of the direct countof basophils and eosinophils. Igakunoayumi 69, 25.

KIMURA, M. & TAKAYA, K. (1979) Ultrastructure ofbasophilic leucocytes and mast cells in normal andcutaneous basophil hypersensitivity-reacted guineapig dermis. Int. Arch. Allergy App. Immunol. 60,303.

RICHERSON, H.B., DVORAK, H.F. & LESKOWITZ, S.(1967) Cutaneous basophil hypersensitivity: A newinterpretation of the Jones-Mote reaction. J. Immu-nol. 103, 1431.

RICHERSON, H.B., DVORAK, H.F. & LESKOWITZ, S(1970) Cutaneous basophil hypersensitivity. I. Anew look at the Jones-Mote reaction, generalcharacteristics. J. exp. Med. 132, 558.

STADECKER, M.J. & LESKOWITZ, S. (1974) The cuta-neous basophil response to mitogen. J. Immunol.113, 496.

WARD, P.A., DVORAK, H.F., COHEN, S., YOSHIDA, T.,DATA, R. & SELVAGIO, S.S. (1975) Chemotaxis ofbasophils by lymphocyte-dependent and lympho-cyte-independent mechanisms. J. Immunol. 114,1523.