Vol. 29, No. 2INFECTION AND IMMUNITY, Aug. 1980, p. 758-7670019-9567/80/08-0758/10$02.00/0

Cutaneous Basophil Hypersensitivity and Contact SensitivityAfter Cutaneous Trichophyton mentagrophytes Infection

FREDERICK GREEN III,' JOHN W. ANDERSON,2 AND EDWARD BALISH3Departments of Surgery,1'3 Anatomy,2 and Medical Microbiology,1 3 University of Wisconsin School of

Medicine, Madison, Wisconsin 53706

The histopathology of cutaneous lesions and trichophytin skin test responseswas examined by light microscopy after the infection of strain 2 guinea pigs withTrichophyton mentagrophytes. Skin biopsies were fixed and stained with proce-dures which allowed differentiation of the polymorphonuclear granulocytic leu-kocytes that were present in lesions or skin test reactions. Basophils comprisedabout one-third of the leukocytes infiltrating the 24 to 48-h trichophytin skin testreactions of guinea pigs sensitized by a cutaneous T. mentagrophytes infection.These results were comparable to the percentage of basophils counted in skintest lesions elicited by the contact agent dinitrochlorobenzene and are consistentwith previously published descriptions of cutaneous basophil hypersensitivity. Incontrast, the active T. mentagrophytes lesion in the skin of guinea pigs sacrificedat defined intervals after infection or reinfection did not appear to containsimilarly elevated numbers of basophils. The early inflammation in primary T.mentagrophytes-induced skin lesions can be characterized histologically as aprimary irritant dermatitis which evolves, during the course of the disease, intoa chronic mononuclear inflammation. This shift apparently results from hostsensitization to fungal antigens during infection and the concurrent developmentof acquired immunity. Reinfection of guinea pigs with T. mentagrophytes resultedin an accelerated cutaneous inflammation that was temporally and histologicallysimilar to allergic contact dermatitis. These results support the hypothesis thatcontact sensitivity to T. mentagrophytes develops during the primary cutaneousinfection of guinea pigs and is an early component of the hypersensitivity responseto reinfection.

The dermatophytes are a group of pathogenicfungi which cause a variety of diseases (tineas)of the skin, hair, and nails in humans and ani-mals. Invasion of the host is limited to the dead,keratinized layers of the epidermis, yet theselocalized infections initiate systemic anamnesticresponses to fungal antigens which are detecta-ble by reinfection, skin testing, and in vitro lym-phocyte transformation (7, 19, 22). Thus, der-matophytosis represents a rather unique infec-tion, and little information is available on themechanisms of immune sensitization and im-mune expression initiated against microbialpathogens multiplying exclusively in dead cuta-neous tissues. The relative roles of acquired an-tibody-mediated immunity or cell-mediated im-munity or both in the pathogenesis and resolu-tion of dermatophytosis are not understood. Formany years cell-mediated immunity has beenassumed to play a primary role in the clearanceof dermatophytosis because individuals sensi-tized by infection with Trichophyton mentagro-phytes manifest a delayed-type hypersensitivity(DTH) to the intracutaneous injection of Tri-chophyton antigen (trichophytin) (15, 20, 38).

This skin test reaction has been assumed to beanalogous to tuberculin hypersensitivity, theprototype of DTH. DTH responses, however,are no longer regarded as a homogeneous groupof reactions (9-11, 13). Several forms of delayed-type cell-mediated hypersensitivity reactions,including contact sensitivity, have recently beencharacterized histologically by an extensive in-filtration of basophilic leukocytes, and these re-actions have been designated cutaneous baso-phil hypersensitivity (CBH) (12, 21).DTH to the intracutaneous injection of tri-

chophytin can be readily elicited in guinea pigssensitized by experimental infection with T.mentagrophytes (6, 7, 22). Tagami et al. (39, 40)examined skin biopsies from humans and guineapigs that were patch tested with trichophytinafter infection with T. mentagrophytes and re-ported histological evidence of allergic contactdermatitis. To date there have been no pub-lished histopathological descriptions of the cel-lular infiltrates in these hypersensitivity reac-tions with fixatives and stains which facilitatethe differentiation of the infiltrating granulo-cytes.

758

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CBH TO T. MENTAGROPHYTES INFECTION

The purpose of this study was to describe thehistopathology of the trichophytin skin test re-action and to compare this reaction with activeprimary and secondary infection lesions withfixation and staining procedures which permitthe differentiation of basophilic leukocytes. Ourresults indicate that trichophytin skin tests inguinea pigs sensitized by infection with T. men-tagrophytes are histologically comparable toCBH. On the other hand, cutaneous reinfectionwith T. mentagrophytes results in a contact hy-persensitivity with few discernible basophils.

MATERIALS AND METHODSAnimals and infection. Strain 2 guinea pigs

weighing at least 300 g were shaved on the flank ordorsal surface and infected by rubbing the hyphae andspores from a 2- to 3-week-old Sabouraud agar plateculture of T. mentagrophytes var. granulosum (ATCC18748) onto the shaved skin with a cotton swab. Atdesignated intervals after infection, animals were ran-domly selected, and biopsy specimens were taken forhistopathological examination.

Trichophytin antigen. T. mentagrophytes wasgrown at 250C in fluid Sabouraud medium (GIBCOLaboratories, Grand Island, N.Y.) on a rotary shaker(150 rpm) for 90 days. A heat-killed soluble antigenwas prepared by autoclaving (15 min, 1210C) andcentrifuging (-350 X g, 10 min) the culture to obtainthe culture supernatant. The protein concentration ofthis supernatant was estimated by the method ofLowry et al. (26).

Skin tests. Guinea pigs were shaved on the flank,and 32 ytg (protein nitrogen) of soluble trichophytin in0.1 ml of pyrogen-free saline (Elkins-Sinn, Cherry Hill,N.J.) was injected intracutaneously. The degree oferythema was scored (0 to 6) as described by Halli-burton and Blazkovec (17). The diameter of erythemawas measured in two planes at right angles to eachother, and the induration (increase in double skinthickness) was assessed with a Schnelltaster (H. T.Kroplin, Hessen, Germany). Measurements weretaken at 30 min, 3 to 6 h, 24 h, and 48 h.

For comparing the histology of dinitrochloroben-zene (DNCB) contact sensitivity with that of tricho-phytin skin tests, guinea pigs were shaved and sensi-tized by the epicutaneous application of 0.01 ml of 1%DNCB (wt/vol) (Sigma Chemical Co., St. Louis, Mo.)in acetone, boosted 2 weeks later with 0.025 ml of 2%DNCB in acetone, and challenged epicutaneously 4weeks later with 0.025 ml of 0.25% DNCB in acetone(10). Erythema and induration were measured as de-scribed above.

Fixation and staining. Guinea pigs were exsan-guinated under anesthesia, and skin reaction sites wereexcised, cut into narrow strips (<5 mm), and fixed inHollande-Bouin fixative (24 to 48 h) (14). Tissues wereembedded in paraffin and sectioned at 5,um. Sequen-tial sections were stained in periodic acid-Schiff todemonstrate fungi or in azure A-eosin B (14) to differ-entiate granulocytic leukocytes. With the latter stain,neutrophils showed many tiny, bright-red granules,eosinophils showed large, fluorescent orange granules,and basophils showed large, oval, pink-purple gran-

ules. Tissue mast cell granules appeared similar incolor to those of the basophilic leukocytes; however,the mast cells were easily differentiated by their largeoval nucleus surrounded by a clear perinuclear cistern.

Cell counts. Differential cell counts were carriedout with a light microscope (oil immersion) by themethod of Dvorak et al. (11). Briefly, cell counts weredone on the leukocytes observed in two selectedswaths (each 100 tym wide) taken vertically throughthe entire thickness of the epidermis and dermis andon the first 200 leukocytes encountered horizontallywithin 100,um of the dermal-epidermal junction. Bothcounting procedures (vertical and horizontal) weredone in the area of the section showing the mostintense infiltration of cells.

RESULTS

Cutaneous hypersensitivity. For describ-ing the temporal development of trichophytinskin test lesions and for differentiating histolog-ically between classical DTH and CBH, guineapigs were skin tested with soluble trichophytinafter the clearance of a primary cutaneous infec-tion with T. mentagrophytes.The gross skin reactions of animals to the

intracutaneous trichophytin injection 8 weeksafter the initiation of active infection with T.mentagrophytes are shown in Table 1. The skintest responses were similar to our previouslypublished results (16) and showed no immediatehypersensitivity during the first 30 min after theintracutaneous injection of the antigen. At 3 h,only 3 out of 11 animals had mild but measurableintermediate erythema. These skin reactionsreached maximum intensity at 24 h and werestill measurable, but reduced, at 48 h.The histopathology of these skin test reaction

sites was examined in skin biopsies taken at 24and 48 h after the intradermal injection of thetrichophytin. Differential cell counts were com-pared with those performed on the inflammatoryinfiltrates of guinea pigs challenged by the epi-cutaneous application of DNCB (Table 2).DNCB skin testing was done to confirm previ-ously reported descriptions of CBH and to verifythe efficacy of our fixation and staining proce-dures. Macroscopic skin reactions after the epi-cutaneous challenge with DNCB were obvious:at 24 h the mean induration and the diameter oferythema were 0.47 ± 0.4 and 21 ± 5 mm,respectively.As shown in Fig. 1, the inflammatory infiltrate

observed after the trichophytin skin test wascharacterized by accumulations of leukocytes inthe upper dermis. Perivascular cuffing withmonocytes was observed in the hypodermis at24 h. Veins within the hypodermis were en-gorged with erythrocytes, indicative of vasodi-latation. Basophilic leukocytes were not evenlydistributed along the upper dermis but were

VOL. 29, 1980 759

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

760 GREEN, ANDERSON, AND BALISH

TABLE 1. Cutaneous hypersensitivity response of strain 2 guinea pigs tested with soluble trichophytin (32pg/O.O1 ml) 8 weeks after a primary cutaneous T. mentagrophytes infection

Skin test responseTime after skin

testing (h) Degee of erythemab Mean diameter of erythema Mef- induration (mm)(mm)

3 0.73 ± 0.79 (11/11) 6.75 ± 1.71 (3/11) 0.45 ± 0.25 (11/11)24 2.64 ± 0.67 (11/11) 11.23 ± 1.9 (11/11) 0.68 ± 0.30 (11/11)48 1.36 ± 0.81 (11/11) 8.59 ± 2.9 (11/11) 0.27 ± 0.23 (11/11)

a Mean ± the standard deviation of positive responders. Numbers in parentheses represent the number ofresponses out of the total number of animals.

b Arbitrarily scored (0, trace; 1, light pink; 2, pink; 3, dark pink; 4, erythema +; 5, erythema ++; 6, hemorrhage)as described by Halliburton and Blaskovec (17).

TABLE 2. Cellular characterization of skin test lesions induced in strain 2 guinea pigs sensitized with T.mentagrophytes or with DNCB

No. of Swath Cells counted'Skin test antigen' ani- Sath

mals sample Baso Eosin MC Neutro Mono Total

Trichophytin (24 h) 3 2V 41 ± 27 6 ± 3 NDd 83 ± 38 153 ± 47 283 ± 84H 66 ± 52 2 ± 2 ND 40 ± 37 95 ± 24 200

Trichophytin (48 h) 3 2V 61 ± 43 14 ± 9 2 ± 2 22 ± 18 302 ± 95 406 ± 116H 64 ± 43 2 ± 3 1 ± 1 1 ± 1 132 ± 45 200

DNCB (24 h) 2 2V 15 ± 4 15 ± 11 2 ± 2 1 ± 1 152 ± 42 184 ± 54H 43±8 10± 13 2± 1 1 1 147±4 200

DNCB (48 h) 2 2V 132 ± 51 35 ± 10 3 ± 2 2 ± 0 242 ± 18 413 ± 40H 63±44 21±9 3±4 0 115±50 200

a Trichophytin, 32 ,ug/O.1 ml; DNCB, 0.25%/0.02 ml.'2V, Total cells encountered in two 100-gm vertical swaths through the epidermis and dermis; H, first 200

cells encountered within 100 gim of the dermal-epidermal junction.'Mean ± the standard deviation. Baso, Basophils; eosin, eosinophils; MC, mast cells; neutro, neutrophils;

mono, mononuclear cells.d ND, Not determined.

clumped in discrete foci (Fig. 2), often surround-ing dilated lymphatic channels. The epidermisexhibited mild acanthosis and spongiosis at 24h. Low numbers of monocytes and neutrophilsand an occasional basophil were observed infil-trating the epidermis. Mononuclear cell countsand total cell counts increased between 24 and48 h (Table 2).

Neutrophils were essentially nonexistent inthe DNCB contact sensitivity reactions at either24 or 48 h, indicating an eliciting dose below thethreshold of toxicity. The DNCB contact reac-

tions were also characterized by the accumula-tion of mononuclear cells and basophils in theupper dermis. Blood vessels in the lower dermiswere dilated and congested without perivascularcuffing. The epidermis was typically edematous,with mild acanthosis and a few mononuclearcells. Basophils constituted 22% of the leuko-cytes in the upper dermis (horizontal countingprocedure) in the DNCB skin tests at 24 h and32% at 48 h. This compares closely with previousdescriptions of CBH responses to contact-sensi-tizing agents in guinea pigs (1, 12, 29).

FIG. 1. Histopathological characteristics of a positive trichophytin skin test at 24 h. A guinea pig was skintested 2 weeks after the resolution of a primary T. mentagrophytes infection. Mild intracellular edema of theepidermis and dense leukocytic infiltration of the upper dermis were present. Azure A-eosin B stain;magnification x200.

FIG. 2. Higher magnification offoci in basophilic leukocytes (arrows) in the upper dermis ofthe trichophytinskin test shown in Fig. 1. Azure A-eosin B stain; magnification x640.

FIG. 3. Histological features of normal, uninfected guinea pig skin. Azure A-eosin B stain, magnificationx200.

FIG. 4. Histopathological characteristics of guinea pig skin 48 h after cutaneous reinfection with T.mentagrophytes. Intracellular edema, infiltration of lymphoid cells into the thickened epidermis, and alymphohistiocytic infiltration of the upper dermis were present. Azure A-eosin B stain; magnification X400.

INFECT. IMMUN .

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CBH TO T. MENTAGROPHYTES INFECTION 761

a ...~~~~~~~~~~h.4K

* )' S tS * R4,. a

*~~~,4, 0',.. _

:,. *. X~~~~

At r

8 + ' '-

S, J , f

I a I* - A

a.. *7.Jes* , s w s~~~~A

P.;.A.

s%

_

'I

p

i-

...42'

. ...~

JSJ

p.0 '*-v.0

4 l

rv f .4*

.,

y. 04/1%x~f4N.

'SZ M 2

, Sjf .. - * .Ks -w w a 4E. _

f vS

.' aZ.e

b

&k N

r0*

I¢

*."

Al" If.i.X!'v4'= F~~~~~~~~~~I1

.fq e i

V

A6 ii .4I> 6 w .

r . d"

_ 1s.^..e-_. ad

r p. r '

v. 4*~4.. * * -._

f'fF-Asi ^

f~~~~ *qpji, 6, 4 of,'4

e

I w~~~~~~~~I

VOL. 29, 1980

rk

4 a4.@I

a

-0

_. p .

41P

10..r .,u

4k. last

FeII

AM.,A-10.1 ....

i

t. 4.,1 w

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

762 GREEN, ANDERSON, AND BALISH

Histology of the primary lesion. For char-acterizing the cellular inflammation of the cu-

taneous, dermatophyte-induced lesions, guineapigs were infected with T. mentagrophytes. Afterinfection with T. mentagrophytes, skin biopsieswere removed at specified intervals for histolog-ical examination and quantitative cell counts(Table 3).During the first 4 days after primary infection,

there were few apparent changes in the histologyof the skin. In normal, uninfected guinea pig skin(Fig. 3) the epidermis was two to four cells thick,and there was only an occasional mononuclearcell or neutrophil in the dermis.The first microscopic evidence of tissue

change appeared on day 5, when moderate num-bers of granulocytes were observed in the der-mis. Increased numbers of eosinophils and a fewfoci of basophils appeared in the upper dermis(day 5), followed by an increase in neutrophilson day 6. In periodic acid-Schiff-stained sectionshyphae were visualized on day 5 throughout the

stratum corneum and penetrating shallow hairfollicles. Neutrophils increased and became thepredominant granulocytic inflammatory cells inthe dermis (day 6 to 7), whereas the number ofeosinophils and basophils decreased (Table 3).Simultaneously, fungal hyphae were found alongthe inner layer of the stratum corneum, and hairfollicles were packed with both hyphae andspores. Infected hair follicles showed less cellularinflammation and abscess formation than thesuperficial epidermis during the early phases ofinfection.One week after infection (day 7), the epidermis

was thickened, and focal accumulations of neu-

trophils in the form of acute microabscessesappeared both within and external to the epi-dermis. Some of the exfoliated neutrophils wereseen in close contact with the invading fungalhyphae in the stratum corneum. Although hy-phae were evenly distributed along the stratumcorneum in some sections, neutrophils were

found in distinct focal abscesses, leaving some

TABLE 3. Cellular characterization of cutaneous lesions during primary T. mentagrophytes infection

Days after No. of Swath Cells counted'infection animals sample' Baso Eosin MC Neutro Mono Total

0 3 2V 0 1±1 1±1 0 198±55 196±35H 0 0 8 ± 3 0 192 ± 3 200

4 2 2V 0 2±7 10±6 2± 1 137±21 150± 13H 1± 1 2± 1 18± 11 1 ± 1 180±9 200

5 2 2V 11±1 47±11 3±4 0 153±12 216±50H 11 ± 7 29 ± 14 5 ± 5 0 155 ± 4 200

6 2 2V 3±4 1±1 3±0 9±1 211±4 281±76H 7 ± 6 4 ± 4 7 ± 6 18 ± 12 164 ± 8 200

7 3 2V 1 ± 1 1 ± 1 NDc 20 ± 23 235 ± 71 257 ± 85H 3 ± 3 6 ± 10 ND 16 ± 12 175 ± 18 200

11 3 2V 10± 8 15 ± 15 ND 86 ± 42 503 ± 236 617 ± 298H 3 ± 5 7 ± 10 ND 17 ± 16 163 ± 29 200

14 3 2V 5 ± 4 6 ± 10 ND 70 ± 59 430 ± 120 514 ± 100H 3 ± 5 7 ± 10 ND 42 ± 37 166 ± 24 200

17 3 2V 6±5 9±8 ND 67±21 398±23 481 ±44H 9±9 7±7 ND 9±4 174± 11 200

21 3 2V 48±53 11±6 4±3 .i 46±50 506±176 624±195H 46 ± 49 2 ± 2 7 ± 8 17 ± 28 127 ± 38 200

28 3 2V 10±7 16±7 5±4 14±23 417±85 462±96H 6±10 9±9 6±7 8±11 170±14 200

a2V, Total cells encountered in two 100-ftm vertical swaths through the epidermis and dermis; H, first 200cells encountered within 100 jAm of the dermal-epidermal junction.'Mean ± the standard deviation. Baso, Basophils; eosin, eosinophils; MC, mast cells; neutro, neutrophils;

mono, mononuclear cells.'ND, Not determined.

INFECT. IMMUN.

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CBH TO T. MENTAGROPHYTES INFECTION

areas of infection free from cellular infiltration.Large neutrophilic epidermal abscesses were

seen on days 9 to 11 (Fig. 5). Epidermal acan-thosis and parakeratosis were prominent, andthe stratum corneum was hyperkeratotic. Thefungi in the outer layers of the stratum corneumappeared to be sloughed in desquamated scales(Fig. 5). Concurrent with the neutrophilic infil-trate in the epidermis the dermis became con-gested with large numbers of mononuclear cells,indicative of a shift to a chronic inflammation.Many of the mononuclear cells were large, witha granular cytoplasm (compatible morphologi-cally with macrophages).On days 11 to 14 superficial ulceration of the

epidermis, with necrosis of tissue in the upperdermis, was visible (Fig. 6). Clumps of pycnoticnuclei were packed into subepidermal areas ofnecrotic exudate. Severe acanthosis of the epi-dermal surfaces adjacent to the areas of ulcera-tion was evident on day 14. Infiltration of thedeep dermis had also increased, and the fattydermis was congested with infiltrating cells, pri-marily mononuclear. Low numbers of fungi per-sisted in the lower layers of the parakeratoticregions, and hyphae and arthrospores were stillobserved in deep hair follicles, some of whichexhibited a neutrophilic folliculitis. Arthrosporeformation appeared limited to hair follicles.By day 17, as healing of the ulceration began,

the epidermis was more parakeratotic, but neu-trophils in microabscesses persisted in the epi-dermis. The upper dermis was hemorrhagic,probably the result of ulcerative erosion, and thehypodermis had become fibrotic. Some extrudedbasophil granules were seen in the papillarydermis below the areas of epidermal ulceration.Relatively few fungal hyphae, some clear andswollen, persisted in hair follicles on day 17.The epidermis remained markedly acanthotic,

with some crusts still attached 3 weeks after theinitiation of infection. Some small focal ab-scesses, primarily neutrophilic, remained in theareas of parakeratosis. Necrotic areas of theupper dermis were in the vicinity of ulcerations.Crusts containing the few remaining observablefungi were being shed at day 21. Fungal hyphaeor spores were no longer visible in hair follicles.In the upper dermis of some day-21 biopsiesthere were focal accumulations of basophils,some partially degranulated, in areas of healingtissue.Four weeks after infection, epidermal lesions

were still acanthotic (seven to twelve cells). Fo-cal parakeratosis with neutrophilic infiltrationpersisted in some regions; however, other areasapproached normal keratinization. Dried scabswere nearly sloughed off, and no fungi werevisible. Although fibrotic and granulocytic tis-

sues provided further evidence of healing in thedermis at day 28, isolated hair follicles were stillundergoing neutrophilic infiltration. Many hairfollicles had lost the hair shaft and had revertedto a rudimentary stage.Histology during reinfection. Secondary

infections were routinely initiated in areas of theskin not corresponding to the primary scar. Onebiopsy (one of three) of skin at day 0 may havebeen from an area of previous T. mentagro-phytes infection because there was a mild tomoderate acanthosis (ca. six cell layers) and anincreased infiltrate of basophils and mast cells(Table 4). All mast cells were clearly granulated.Macroscopically, 48 h after reinfection, a few

cutaneous papules of mild to moderate erythemawere visible. Microscopically, the cellular infil-tration was of variable intensity. The epidermisof some animals was undergoing mild, focalacanthosis and spongiosis, with small mononu-clear cells and neutrophils migrating into theareas where hyphae and spores were seen on thestratum corneum (Fig. 4). In other 48-h lesionbiopsies, dermal microabscesses (neutrophilic)and focal ulceration of the epidermis were pres-ent in the areas where fungi were observed inthe stratum corneum. This accelerated an-amnestic response is temporally and histologi-cally similar to cell-mediated allergic contactsensitivity. Unlike the contact sensitivity elicitedby DNCB, however, only a few basophils wereobserved in the 48-h reinfection inflammatoryresponse (Table 4).One week after reinfection (day 7), massive

acanthosis and focal parakeratosis could be seenin the areas adjacent to necrotic epidermal neu-trophilic abscesses. The cellularity of the hypo-dermis was mild and diffuse, with few neutro-phils or plasma cells. In contrast to the profusefungal invasion seen at day 7 of a primary infec-tion, few fungal hyphae were seen in the stratumcorneum and in hair follicles. Few day-14 lesionscontained visible hyphae, and by day 21, nofungal hyphae or spores were visible.A comparison of the cellular infiltrates during

primary infection (Table 3) with those observedafter reinfection (Table 4) demonstrates thatthere were fewer inflammatory cells at the peakof reinfection (day 7) than at the peak of theprimary infection (day 11). This accords withthe reduced severity and more rapid resolutionof the lesions observed macroscopically duringreinfection.

DISCUSSIONThe observation of CBH in biopsies of tricho-

phytin skin tests in guinea pigs sensitized byinfection suggested that active reinfection le-sions would also exhibit CBH. A major result of

763VOL. 29, 1980

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

.yD

' ; J

,

pIr

r.s.lbtaC' ^,-.-

'.4

I

r *. .**/r K. I.- 9

/ A

>r.< .; *a C .

.A,,,irZ'

i

p

2>AtI

I-.

...

.

.

..., I

. ..

No

lk1w

nw.a

FIG. 5. Histopathology of T. mentagrophytes-infected skin (day 11 postinfection). Fungal hyphae u'erepacked in the stratum corneum andpenetrating hair follicles (arrow). A neutrophilic microabscess containingnecrotic debris can be seen within the parakeratotic epidermis. Periodic acid-Schiff stain; mangnificationx155.

FIG. 6. Histopathological characteristics ofa cutaneous T. mentagrophytes infection (day 11 postinfection).The epidermis was ulcerated (arrow) and necrotic. Dense lymphohistiocytic and neutrophilic infiltration werepresent in the dermis. Fungal hyphae were visible in the stratum corneum above the intact epidermis. Periodicacid-Schiff stain; magnification xI 75.

764

ft00A. -i,

......

4e V

.1

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CBH TO T. MENTAGROPHYTES INFECTION 765

TABLE 4. Cellular characterization of cutaneous lesions during reinfection with T. mentagrophytesAd -t a_C STY _£ 2__@AzICells counted'Days after No. of

_ Dortini AM AIwCPA__SwathO mnpL

Baso Eosin MC Neutro Mono Total

0 3 2V 2+2 0 11±11 0 223±48 238±55H 17 ± 26 2 ± 4 11 ± 7 0 169 ± 24 200

2 3 2V 9±5 9±8 5±3 65±59 183±63 304±86H 7 ± 3 4 ± 3 4 ± 5 44 ± 37 142 ± 33 200

7 5 2V 10±7 3±3 5±2 91±72 284±84 408±142H 9 ± 5 4 ± 7 3 ± 3 40 ± 29 144 ± 26 200

14 6 2V 5±9 4±5 2±2 63±48 325±79 397±91H 7 ± 7 9 ± 11 3 ± 2 16 ± 12 149 ± 33 200

21 4 2V 1 ± 1 12± 10 7±8 13± 14 339±92 371 ± 107H 1 ± 2 6 ± 7 8 ± 7 2 ± 3 183 ± 4 200

a 2V, Total cells encountered in two 100-,Um vertical swaths through the epidermis and dermis; H, first 200cells encountered within 100 Am of the dermal-epidermal junction.

b Mean ± the standard deviation. Baso, Basophils; eosin, eosinophils; MC, mast cells; neutro, neutrophils;mono, mononuclear cells.

this study was the failure to observe CBH in T.mentagrophytes reinfection lesion biopsies. Thisapparent paradox in the absence of focal accu-mulations of basophils in lesion biopsies takenduring infection may be the result of the follow-ing: (i) selective differences between the anti-genic compositions of the heat-killed soluble an-tigen and the growing fungi, (ii) rapid degranu-lation of basophils chemotactically summonedinto the lesions (3, 8, 36), or (iii) suppression ofCBH by DTH, as reported by Askenase (2).Current data do not permit the exclusion of anyof these hypotheses; however, many of the ba-sophils identified in infected lesion biopsies ap-peared to be in stages of degranulation. Thus,there could have been undetected basophils inthese tissues, consistent with hypothesis iiabove.Although reinfection lesions do not contain

elevated levels of basophils, they do manifestseveral other histological features of contact sen-sitivity 48 h after reinfection, i.e., spongiosis,acanthosis, and parakeratosis. Other evidencesupporting the concept of an intimate associa-tion between contact sensitivity and cutaneousT. mentagrophytes infection includes the follow-ing: (i) the unique extracellular location offungalreplication during infection (restricted to thedead, keratinized layers of the skin and hairfollicles); (ii) the accelerated macroscopic in-flammatory response elicited 24 to 48 h after thecutaneous reinfection of guinea pigs sensitizedby active infection (16, 22); and (iii) the positivetrichophytin contact sensitivity patch testing ofhumans and guinea pigs after active infection(39, 40).

Histopathologically, the trichophytin skin

tests are not identical with DNCB contact sen-sitivity; however, one cannot simply comparethe epicutaneous application ofa skin-sensitizinghapten (DNCB) with the intradermal injectionof a mosaic of fungal antigens (soluble tricho-phytin). Trichophytin skin test lesions containedmany neutrophils, indicating either an overlap-ping antibody-mediated hypersensitivity (5) ora severe necrotic reaction, whereas DNCB skintests contained essentially no neutrophils and amodest increase in eosinophils, as described byAskenase (1).

Reports of basophilic infiltration of cutaneoustissue after microbial infection are few (4, 36).Sohnle and Kirkpatrick reported the presence ofinflammatory foci of basophils along the dermalbasement membranes of skin during cutaneouscandidiasis in guinea pigs (36); however, theydid not characterize the histology of skin testresponses to Candida antigen in sensitized ani-mals. Their studies concluded that the basophilaccumulations were due to a mechanism inde-pendent of antibody-mediated immunity, cell-mediated immunity, or direct chemotaxis toCandida antigen or complement (36). The con-clusions of Sohnle and Kirkpatrick may havebeen confounded by the rapid growth rate of C.albicans under occlusive dressings which resultsin secondary infections that are neither shorternor more intense than the primary infections(28, 35).The histopathological results presented in this

study indicate that the response of guinea pigsto a primary cutaneous infection with T. men-tagrophytes can be characterized as an exfolia-tive dermatitis which appears to progress fromacute to chronic. The early inflammation during

VOL. 29, 1980

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

766 GREEN, ANDERSON, AND BALISH

the primary infection closely compares with de-scriptions of primary irritant dermatitis (29), thesalient feature of which is necrosis of the epider-mis and the upper dermis (23) which may beinitiated by the production of toxic products bythe fungi (25, 31). A similar histopathologicalpicture is observed in guinea pig skin whenDNCB in toxic concentrations (exceeding 1%) isapplied to the skin of nonsensitized animals (29).Contact sensitivity to DNCB administered atlevels below the toxic threshold can be elicitedas early as day 4 post-sensitization (33), andareas of severe epidermal necrosis are invadedby neutrophils (10). Therefore, the presence ofneutrophils during primary T. mentagrophytesinfection (days 5 to 9) could also be attributedto early tissue damage caused by the expressionof contact hypersensitivity (38). Because neithergranulocytes nor monocytes were observed inphysical contact with hyphae in lesion biopsies,the elimination of the fungi may be mediated bysoluble fungistatic serum mediators, as postu-lated by King et al. (24). Contact sensitivitywould serve to damage the epithelial barrier,causing the release of fungistatic or fungicidalfactors (from serum or cells). Contact sensitivityis thought to be mediated by the interaction ofperipheral T-lymphocytes with soluble foreignantigens which diffuse into viable tissues (27, 30,37). This mechanism could also provide a rapidanamnestic recognition of a microbial invasionof the keratinized layers of the skin. Theoreti-cally, subsequent chemotaxis and degranulationof basophils (CBH) could enhance the evolutionof the host's inflammatory response to derma-tophytosis (32). Thus, contact sensitivity andCBH may precede DTH in response to replicat-ing microbial antigens in the skin and as suchwould not be mutually exclusive but would besynergistic expressions of cell-mediated immu-nity to superficial skin infections. Conversely,contact sensitivity and CBH have been postu-lated as examples of T-cell-mediated reactionsmodulated by B-cell function (33, 34, 41). Sup-pression of the hypersensitivity to dermatophyteantigens could be an important mechanism forresolving cutaneous T. mentagrophytes infec-tions and limiting tissue damage (16, 42).The definitive relationship of a CBH-like tri-

chophytin skin test response and contact sensi-tivity to the pathogenesis, resolution, or immunemodulation of dermatophyte infections or all ofthese will require further investigation. It is ofinterest to note that trichophytin, Candida an-tigen, and DNCB are routinely used to evaluatethe cell-mediated immunity competence of can-cer patients in vivo (18). The absence of a meas-urable response to these antigens might reflect

INFECT. IMMUN.

a defect in CBH rather than an anergy to clas-sical DTH.

ACKNOWLEDGMENTS

We acknowledge the technical assistance of Celia Evers inthe preparation of histological sections.

This study was supported in part by grants from the U.S.Army Medical Research and Development Command(DAMD 17-75-C-5004) and the National Institute of Allergyand Infectious Diseases (AI-15119).

LITERATURE CITED

1. Askenase, P. 1973. Cutaneous basophil hypersensitivityin contact-sensitized guinea pigs. J. Exp. Med. 138:1144-1155.

2. Askenase, P. W. 1977. Role of basophils, mast cells andvasoamines in hypersensitivity reactions with a delayedtime course. Prog. Allergy 23:199-320.

3. Askenase, P. W., R. Debernardo, D. Tauben, and M.Kashgarian. 1978. Cutaneous basophil anaphylaxis.Immunology 35:741-755.

4. Clark, J. M., G. Altman, and F. B. Fromowitz. 1977.Basophil hypersensitivity response in rabbits. Infect.Immun. 15:305-312.

5. Colvin, R. B., V. W. Pinn, B. A. Simpson, and H. F.Dvorak. 1973. Cutaneous basophil hypersensitivity. IV.The late reaction. J. Immunol. 110:1279-1289.

6. Cruickshank, D. N. D., M. D. Trotter, and S. R. Wood.1960. Studies on trichophytin sensitivity. J. Invest. Der-matol. 35:219-223.

7. DeLamater, E. D., and R. W. Benham. 1938. Experi-mental studies with the dermatophytes. II. Immunityand hypersensitivity produced in laboratory animals. J.Invest. Dermatol. 1:469-488.

8. Dvorak, A. M., M. C. Mihm, and H. F. Dvorak. 1976.Degranulation of basophilic leukocytes in allergic con-tact dermatitis reactions in man. J. Immunol. 116:687-695.

9. Dvorak, H. F. 1974. Delayed hypersensitivity, p. 291-345.In B. W. Zweifach et al. (ed.), The inflammatory proc-ess, vol. 3. Academic Press, Inc., New York.

10. Dvorak, H. F., A. M. Dvorak, and M. C. Mihm. 1974.Morphologic studies in cellular hypersensitivity inguinea pigs and man. Monogr. Allergy 8:54-65.

11. Dvorak, H. F., A. M. Dvorak, B. A. Simpson, H. B.Richardson, S. Leskowitz, and M. J. Karnovsky.1971. Cutaneous basophil hypersensitivity. II. A lightand electron microscopic description. J. Exp. Med. 132:558-582.

12. Dvorak, H. F., and M. Mihm. 1972. Basophilic leuko-cytes in allergic contact dermatitis. J. Exp. Med. 135:235-254.

13. Dvorak, H. F., B. A. Simpson, R. C. Bast, and S.Leskowitz. 1971. Cutaneous basophil hypersensitivity.III. Participation of the basophil in hypersensitivity toantigen-antibody complexes, delayed hypersensitivityand contact allergy. Passive Transfer. J. Immunol. 107:138-148.

14. Fullmer, H. M., and R. D. Lillie. 1976. Histopathologictechnic and practical histochemistry, 4th ed. The Blak-iston Division, McGraw-Hill Book Co., New York.

15. Grappel, S. F., C. T. Bishop, and F. Blank. 1974.Immunology of dermatophytes and dermatophytosis.Bacteriol. Rev. 38:222-250.

16. Green, F., III, and E. Balish. 1979. Suppression of invitro lymphocyte transformation during an experimen-tal dermatophyte infection. Infect. Immun. 26:554-562.

17. Halliburton, B. L., and A. A. Blazkovec. 1975. Delayedhypersensitivity and acquired cellular resistance inguinea pigs infected with Listeria monocytogenes. In-fect. Immun. 11:1-7.

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CBH TO T. MENTAGROPHYTES INFECTION 767

18. Hamilton, D. N. H., V. Ledger, and A. Diamanopour-ous. 1976. In vitro monitoring of cell-mediated immu-nity to dinitrochlorobenzene. Lancet ii: 1170-1171.

19. Hunjan, B. S., and L. S. Cronholm. 1978. An animalmodel for cell-mediated immune responses to derma-tophytes. J. Allergy Clin. Immunol. 63:361-369.

20. Jones, H. E., J. H. Reinhardt, and M. G. Rinaldi. 1974.Model dermatophytosis in naturally infected subjects.Arch. Dermatol. 110:369-374.

21. Katz, S. I., C. J. Heather, D. Parker, and J. L. Turk.1974. Basophilic leukocytes in delayed hypersensitivityreactions. J. Immunol. 113:1073-1078.

22. Kerbs, S., J. Greenberg, and K. Jesrani. 1977. Tem-poral correlation of lymphocyte blastogenesis, skin testresponses and erythema during dermatophyte infec-tions. Clin. Exp. Immunol. 27:526-530.

23. Kerl, H., G. Berg, and 0. Braun-Falco. 1974. Quanti-tative and qualitative dynamics of the epidermal andcellular inflammatory reaction in primary toxic andallergic dinitrochlorobenzene contact dermatitis inguinea pigs. Arch. Dermatol. Forsch. 249:207-226.

24. King, R. D., H. A. Khan, J. C. Foye, J. H. Greenberg,and H. E. Jones. 1975. Transferrin, iron and derma-tophytes. I. Serum inhibitory component definitivelyidentified as unsaturated transferrin. J. Lab. Clin. Med.86:204-212.

25. Kligman, A. M. 1955. Tinea capitis due to M. audouiniiand M. canis. Arch. Dermatol. 71:313-337.

26. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

27. Macher, E., and M. W. Chase. 1968. Studies on thesensitization of animals with simple chemical com-pounds. XI. The fate of labeled picryl chloride anddinitrochlorobenzene after sensitizing infections. J. Exp.Med. 129:81-121.

28. Maibach, H., and A. Kligman. 1962. The biology ofexperimental human cutaneous moniliasis (Candidaalbicans). Arch. Dermatol. 85:233-267.

29. Medenica, M., and A. Rostenberg. 1971. A comparativelight and electron microscopic study of primary irritantcontact dermatitis and allergic contact dermatitis. J.Invest. Dermatol. 56:259-271.

30. Medewar, P. B. 1965. Transplantation of tissues andorgans: introduction. Br. Med. Bull. 21:97.

31. Minocha, V., J. S. Pasricha, L. N. Mohaptra, and K.C. Kandhari. 1972. Proteolyte activity of dermato-phytes and its role in the pathogenesis of skin lesions.Sabouraudia 10:79-85.

32. Pearson, D. J., and G. Taylor. 1977. Augmented localanaphylaxis at sites of delayed hypersensitivity skinreactions. Immunology 33:115-119.

33. Polak, L., and C. Rinck. 1977. Effect of elimination ofsuppressor cells on the development of DNCB contactsensitivity in guinea pigs. Immunology 33:305-311.

34. Rocklin, R. E. 1976. Modulation of cellular-immune re-sponses in vivo and in vitro by histamine receptor-bearing lymphocytes. J. Clin. Invest. 57:1051-1058.

35. Sohnle, P. G., M. M. Frank, and C. H. Kirkpatrick.1976. Mechanisms involved in elimination of organismsfrom experimental cutaneous Candida albicans infec-tion of guinea pigs. J. Immunol. 117:523-530.

36. Sohnle, P. G., and C. H. Kirkpatrick. 1977. Study ofpossible mechanisms of basophil accumulation in ex-perimental cutaneous candidiasis in guinea pigs. J. Al-lergy Clin. Immunol. 59:171-177.

37. Streilein, J. 1978. Lymphocyte traffic, T-cell malignan-cies and the skin. J. Invest. Dermatol. 71:167-171.

38. Sulzberger, M. B. 1949. Immunologic changes broughtabout by fungi and fungus products. Ann. N.Y. Acad.Sci. 50:767-772.

39. Tagami, H., S. Watanabe, and S. Ofuji. 1973. Tricho-phytin contact sensitivity in guinea pigs with experi-mental dermatophytosis induced by a new inoculationmethod. J. Invest. Dermatol. 61:237-241.

40. Tagami, H., S. Watanabe, S. Ofuji, and K. Minami.1977. Trichophytin contact sensitivity in patients withdermatophytosis. Arch. Dermatol. 113:1409-1414.

41. Turk, J. L., and D. Parker. 1973. Further studies on B-lymphocyte suppression, indicating a possible mecha-nism for Jones-Mote hypersensitivity. Immunology 24:751-758.

42. Turk, J. L., L. Polak, and D. Parker. 1976. Controlmechanisms in delayed hypersensitivity. Br. Med. Bull.32:165-170.

VOL. 29, 1980

on Decem

ber 23, 2020 by guesthttp://iai.asm

.org/D

ownloaded from