mycoses 31, No.

3 (1988) Effect of Trichophytin and Candidin on Chemotaxis 137

I mycoses 31 (3) 137-142 accepted/angenommen: Januar 16,1987 . 0Grosse Verlag Berlin 1987

The Effect of Trichophytin and Candidin

on Neutrophil Chemotaxis
Die Wirkung von Trichophytin und Candidin
auf die Neutrophilen-Chemotaxis
Vera Leibovici', Ruth Evron' and Y. Matzner2
Department of Dermatology,
Department of Hematology,
Hadassah Ein Karem and Mount Scopus University Hospitals, Jerusalem, Israel

Key words: Trichophytin - candidin - chemotaxis - neutrophils

Schliisselworter:Trichophytin - Candidin - Chemotaxis - Neutrophile

Summary:The antigenicextracts of Trichophy- areas; thus, it constitutes an essentialearly step

ton and to a lesser extent of Cmdiduwere found in the mobilization of cells against an infecting
to inhibit the directed locomotion of polymor- agent (14). It is possible that the failure of
phonuclear leukocytes towards zymosan- neutrophil migration in fungal infections
activated serum. The inhibitory potential was could be one of the causes for the protracted
cell-directed rather than chemotactic factor- course and high incidence of recurrencies in
directed. The fungal antigens did not affect the this type of infection.
neutrophil spontaneous migration. The results Several cutaneous disorders, either ac-
may explain, at least in part, the lack of quired or congenital, have been described in
neutrophilic infiltration in certain fungal association with defects in neutrophil chemo-
infections. taxis (1). Excluding rare congenital disorders,
most of the neutrophil chemotactic impair-
Zusammenfassung: Antigenextrakte von Tri- ments are acquired through malignancies,
chophyton und - in geringerem AusmaD - von diabetes mellitus, liver and autoimmune
Can& hemmen die gerichtete Bewegung diseases, drugs such as colchicin (13), heparin
polymorphkerniger Leukozyten gegenuber (2), aspirin (12) and anti-fungal drugs (9).The
Zymosan-aktiviertem Serum. Das Hemmpo- effect of fungi on neutrophil migration has
tential war eher gegen die Zellen als gegen den been subject to controversial interpretation.
chemotaktischen Faktor gerichtet. Die Pilzan- Moreover, it is doubtful whether neutrophils
tigene beeinflaten nicht die Spontanbeweglich- play any significant role in containing fungal
keit der Neutrophilen. Die Ergebnisse konnten invasion.
- wenigstens teilweise - das Nichtvorhanden- The clinical manifestations of dermato-
sein einer Neutrophilen-Infdtration bei gewis- phytes, which invade and parasitize kera-
sen Pilzinfektionen erklaren. tinized layers of the skin, nail and hair includea
large variety of immune responses to antigenic
Leukocyte chemotaxis is defined as the components of dermatophytes (1, 3). Infec-
directed migration of polymorphonuclear tions with virulent strains of an anthropo-
leukocytes and mononuclear cells toward an zoophilic species, such as Trichophyton
infecting organism or its mediators in inflamed mentagrophytes, result in acute local inflamm-
138 Vera Leibovici et al. mycoses 31, No. 3 (1988)

ation followed by regional lymphadenitis that ground up to pass through a 27 gauge needle.
usually heals spontaneously within three Suspensions of the fungi in absolute ethyl
months. Reinfection results in a highly alcohol were dried in a vacuum oven at 60"C,
accelerated but less severe course of disease. weighed and re-suspended in 0.5% phenol
Although resistance to local reinfection has saline to contain 0.1 mg dried fungus per ml
been demonstrated, tissue immunity exists phenol saline. Phenol was removed using
only at previously infected sites and not at the diaysis against PBS and the antigen solutions
distant skin areas (3). On the other hand, an were stored at 4°C. Final dilutions were
anthropophilic species, such as Trichophyton carried out before use, using PBS at pH 7.4.
rubrum, usually causes more chronic, less Human neutrophils were obtained from
circumscribed infections which result in lower healthy donors and were prepared as
resistance to reinfection. Intradermal injection previously described (2) by dextran sedimen-
of Trichophytin (the antigenic extract of tation, followed by hypotonic lysis and
Trichophyton species) in humans induces a centrifugation over Ficoll-Hypaque (Phar-
hypersentitivity reaction which may be of the macia, Uppsala, Sweden). The cells (> 95%
urticaria1 immediate-type indicating presence neutrophils) were suspended at 106/ml in
ofcirculating antibodies or a retarded delayed- PBS'. Foreachexperiment neutrophilsfrom a
type, generally associated with a cellular different healthy donor were used. Trypan-
immune reaction and resistance to reinfec- blue exclusion was unaffected by treatment
tion. with the fungal antigens.
The lack of neutrophilic infiltration in the Neutrophil migration was measured in
above host-parasite responses is striking. A Boyden chambers using zymosan activated
possible explanation for this phenomenon serum (ZAS)as chemoattractant as previously
could be an anti-chemotactic effect of described (11).Chemotaxis was assayed by the
dermatophytes. The inhibition of neutrophil leading-front technique (11). In the assays the
migration could explain, at least in part, their neutrophil suspension was placed in the upper
relatively minor role in controlling dermato- compartment of the Boyden chamber and the
phyte infection. In the present study we chemoattractant (PBS' containing 1%, v/v
describe the effect of antigenic extracts of ZAS) in the lower compartment. Incubations
Candida and Trichophyton species on were carried out at 37 "C for 45 min. The effect
neutrophil chemotaxis, and demonstrate their of trichophytin or Candidin on neutrophil
inhibitory potential on cell migration. random and directed migration was measured
by incubating them with either the cells in the
upper compartment ofthe Boyden chamber or
Materials and Methods
the chemoattractant in the lower compart-
Blind well Boyden chambers were purchased ment. Incubations were carried out for 10 min
from Nucleopore Corp., Bethesda, Md., and at 37 "C before the chemotaxis assay, after
corresponding filters (Membradilter, 3 mi- which the antigen remained in the chamber
cron pore size) from Sartorius, Gottingen, during the experiment. Results were always
West Germany. The buffer used (PBS') was compared with the normal migration in the
Dulbecco's phosphate buffer (pH 7.4) contain- absence ofthe antigen. Random migration was
ing lmg/ml glucose and 6mg/ml bovine determined by measuring migration against
serum albumin. The fungal antigens were PBS+ alone. Chemotaxis was calculated as the
candidin extracted from C. albicans and distance in microns (p) travelled by the
trichophytin extracted from a 1:l mixture of neutrophils in response to the chemoattrac-
Tmentagrophytes and Trubrum as previously tant minus the random migration. Results
described (10). Briefly, yeast-like fungi were were expressed as the mean f l S E of ten
grown on Sabouraud dextrose agar (Difco), experiments. The statistical sigdicance of the
washed off with absolute ethyl alcohol and results was determined by the paired t-test.
mycoses 31, No. 3 (1988) Effect of Trichophytin and Candidin on Chemotaxis 139

Table 1: The effect of trichophytin and candidin on inhibitory potential was aimed at the
neutrophil chemotaxis toward ZAS neutrophil directed locomotion machinery.
Chemotaxis km
The inhibitory effect of the fungal antigens
Antigen Place of the antigen on neutrophil directed migration was propor-
Uooer chamber Lower Chamber tional to their concentration in the cell
Trichophytin 12.2*6.a* 36.0i6.2 suspension in the upper compartment. Again,
Candidin ia.4ia.7** 33.4i5.1 the inhibitory effect of trichophytin (Fig. 1)
None 36.8~9 36.ai4.9
seemed to be more significant than that of
* p>o.o1 candidin (Fig. 2), especially when the higher
** pi0.05
concentration of fungal antigen was used
Chemotaxis was measured as described in material
and methods using 1 % ZAS (v/v) as chemoattractant.
Trichophytin or candidin (lo-' v/v) were placed in
either the upper compartment with neutrophils or in Discussion
the lower compartment with the chemoattractant.
Results were corrected for random migration of In this study we describe the inhibitory effect of
52.5f2.7pm and are expressed as the mean & 1 SE of fungal antigens on neutrophil chemotaxis in
10 experiments.
vitro. The mechanism is not yet understood,
but the inhibitory activity seems to affect the
Table 2: The effect of trichophytin and candidin on cells directly. Since its effect could be
random migration of neutrophils demonstrated only when the neutrophils were
Chemotaxis (pm) incubated with the antigen, 0.1% antigen
Antigen Place of the Antigen dilutions had the strongest effect, trichophytin
Upper chamber Lower chamber being somewhat more active, and inhibition
Trichophytin 52.9-12.33 51.0k2.93 decreased when lower concentrations were
Candidin 46.6ii .oa 54.8i3.04
None 52.4i2.66 52.4-12.66 used.
It is known that in a'cute infections with
Random migration was assayed after addition of
trichophytin or candidin (lo-'% v/v) either to the dermatophytes of zoophilic origin such as
neutrophils in the upper compartment or the PBS' in Trichophyton verrucosum and Trichophyton
the lower compartment. Results are expressed as the mentagrophytes the appearance of neutrophil
mean f 1 SE of 10 experiments.
containing pustules is common. However
when the skin is infected with antropophilic
species such as Trichophyton rubrum or even
with mixed infections including both species,
onlyfew orno pustules develop(4).Daviesand
Trichophytin and candidin expressed a Zaini (4) investigated the chemotaxis of
substantial inhibitory effect on neutrophil neutrophils toward antigens prepared and
migration toward ZAS when placed in the dissolved in Ringer-Locke solution including
upper compartment of the Boyden chamber 10% autologous plasma and found that this
(Table I). The inhibitory effect of trichophytin mixture contained chemotactic activity.
seemed to be more significant than that of However, when plasma was either omitted
candidin. However, incubation of the fungal from the fungal suspension or heated at 56"C,
antigens with the chemoattractant, did not no neutrophil migration occurred. They
affect neutrophil migration, indicating a cell- concluded that the fungal antigen acted upon
directed rather than chemotactic factor- the plasma to produce a neutrophil chemotac-
directed inhibitory potential. tic factor, probably by activation of the
No inhibitory effect could be demonstrated complement cascade to produce the chemo-
when neutrophil random migration was tactic peptide CSa.
studied following addition of the fungal Our in vitro results seem to be in agreement
antigen to each of the two compartments with similar observations reported by Balogh
(Table 2). T h s suggested that the main et al. (6,7)as well as with clinical observations
140 Vera Leibovici et al. mycoses 31, No. 3 (1988)

I I I
loo3 10-2 10"
T RIC HOPHYT IN C 0 NC ENT RAT 10N
(O/O VIV)
Fig. 1 : The effect of various concentrations of trichophytin on neutrophil chemotaxis.
Chemotaxiswasmeasuredasdescribedusing1%(v/v)ZASaschemoattractant.Trichophytinwasadded to theneutrophils
in the upper compartmentat the concentrations indicated. Results were corrected for random migration of 52.4 f2.7pn-1
and are expressed as the mean + 1 SE of 10 experiments.
P <0.01 and P i O . 0 5 for trichophytin concentrations of lo-' and lo-'% v/v respectively.

of fungal infections in which only a few fungi on humoral factors (other than C,, that
pustules are present (4). The mechanism by was not directly neutralized (in our in vitro
which neutrophil migration toward those studies) on the skin directly, need further
superficial infections is inhibited is probably investigation.Thus, we believe that our in vitro
complicated and constitutes many different studies support in part the hypothesis that
factors. Our results, showing direct inhibition fungal antigens incubated may block one or
of neutrophils by the fungal antigens, may more of the complicated cellular mechanisms
explain one possible mechanism. Other involved in the cell-directed locomotion of
mechanisms, such as hypothetical effect of the neutrophils in superficial mycotic infections.
mycoses 31, No. 3 (1988) Effect of Trichophytin and Candidin on Chemotaxis 141

40

30

20

10

I I // I

lo-'
CAND DIN CONCENTRATION
( O/O V I V )
Fig. 2: The effect of various concentrations of candidin on neutrophil chemotaxis.
The method used is described in Fig. 1.Candidin was added to the neutrophil cell suspension in the upper compartment of
+
the Boyden chamber at the concentrations indicated. Results were correctedfor the random migration of 52.4 2.7 pm and
are expressed as the mean + 1 SE of 10 experiments.
P<O.O5 for lo-' and v/v of candidin concentrations.

References
4. Davies, R. R. and F. Zaini (1984):Trichophyton rubrum
1. Dahl, V. M. (1978):Chemotaxis in cutaneous disease. and chemotaxis of polymorphonuclear leukocytes.
Int. J. Dermatol. 17,95-104. Sabouraudia (J. Med. Veter. Mycol.) 22, 65-71.
2. Matmer, Y., G. Marx, R. Drexler and A. Eldor (1984): 5. Denning, T. J. V. and R. R. Davies (1973): Candida
The inhibitory effect of heparin and related albicans and the chemotaxis of polymorphonuclear
glycosaminoglycans on neutrophil chemotaxis. neutrophils. Sabouraudia (J. Med. Veter. Mycol.) 11,
Thromb-Haemostas. Stuttgart, 52, 134-137. 210-221.
3. Grappel, S. F., C. T. Bishop, F. Blank (1974): 6. Balogh, E., E. Forize, M. Debreczeni, M. Szabolcsy
Immunology of dermatophytes and dermatophytosis. (1981): Serum IgE level and T-cell count in chronic
Bacteriol. Revue 38, 222-250. dermatophytosis. Mykosen 24,84-89.
142 Vera Leibovici et al. mycoses 31, No. 3 (1988)

7. Balogh, E., E. Forizs and M. Debreczeni (1985): 12. Matzner, Y.,R.Drexler and M. Levy (1983): The effect
Investigations on the chemotactic and phagocytic of dipyrone, aspirin and paracetamol on human
activities of circulating monocytes in chronic chemotaxis. Eur. J. Clin. Invest. 14,440-442.
dermatophytosis. Mykosen 28,490-494. 13. Phelps, P.(1970):Appearance of chemotactic activity
8. Ottaviano, P. J., H. E. Jones, J. Jaeger, D. R. King and followingintracellular injection of monosodium urate
D. Bibel (1974): Trichophyton extraction. Biological crystals: Effect of colchicin. J. Lab. Clin. Med. 76,
comparison of trichophytin extracted from Trichophy- 622-624.
ton rnentagrophytes grown in a complex medium or a 14. Senn, H. G. and W. S. Fungi (1975): Neutrophil
defined medium. Appl. Microbiol. 28,271-275. migration in health and disease. Semin. Hematol. 12,
9. Davies, R. R. and F. Zaini (1985): Antifungal drugs 27-45.
affecting the chemotaxis of polymorphonuclear
neutrophils. Sabouraudia (J. Med. Veter. Mycol.) 23,
119-123.
10. Beemer, A. M., E. S . Kuttin and M. Pinto (1977):
Treatment with antifungalvaccines. Contr. Microbiol.
Immunol. 4, 136- 146. Address: Dr. Vera Leibovici,
1 1 . Matzner, Y.,R. E. H. Partridge and B. M. Babior Department of Dermatology,
(1983): Chemotactic inhibitor in synovial fluid. Hadassah University Hospital,
Immunology 49, 131-138. P.O.B. 12018, Jerusalem 91 120, Israel

Miscellaneous

Eduard Grosse Senior Awards

to Munich, Gottingen and Dusseldorf

Berlin

The Eduard Grosse Award, named for the International Journal of Andrology”. The
founder of Grosse Publishing Company, will authors are W. Engel and S. Florke-Gerloff of
be conveyed yearly as of 1988 for understand- the Institute for Human Genetics, Gottingen,
ing and unusual work in research as well as and E. Topfer-Petersen, W.-B. Schill of the
therapy. Dermatological Clinic of Munich University.

This year’s selected works, published in 1987in As thebest workin thefieldoftherapy., thejury
the company’sjournals, were chosen by a jury chose “Clinic and Differential Diagnosis of
comprised of the editors of the magazines Cutaneous Lupus Erythematosus” by P. Kind
“H + G Zeitschrift fur Hautkrankheiten”, and G. Goerz of the University-Skin Clinic of
“mycoses”, “andrologia” and “Der Deutsche Diisseldorf. It was published in “H + G , issue
Dermatologe”. The foreman of the jury was 18/87.
G. K. Steigleder, Cologne.
The awards will be presented to the recipients
The best scientific work, “Evolution and during the 35th Meeting of the German
Development in the Outer Acrosomal Dermatological Society in Munich in April.
Membrane (OAM) and Evidence that The Eduard Grosse Senior Award was
Acrosin-Inhibitors are Proteins OAM” was established in 1986 on the 40th anniversary of
published in issue 2/87 of “andrologia First Grosse Publishing Company in Berlin.