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An animal model for cell-mediated immune responses to dermatophytes
An animal model for cell-mediated immune responses to dermatophytes B. S. Hunjan, Louisville,
Ph.D., and L. S. Cronholm,
Ph.D. Chandigarh, India,
and
KY.
Guinea pigs in&ted with three species of dermatophytes were tested for cell-mediated immune responses, incYuding delayed cutuneous hypersen.~itiL’ity. (w~phocyte transf&mation (LT), muc,rophuge migration inhibition (MMIJ. and passive tran?fer of hypersensitivity. The patterns of reinfection disease were also unalyzed as an index of altered host immunity. The fungi tested represented the three genera oj’derrnatoph~tes (Trichophyton, Microsporum, and Epidennophyton), and included zoophilic and anthropophilic, species. Microsporum vanbreuseghemii, the zoophilic species, produced the most virulent infection, and Epidemwphyton floccosum, previously considered obligutely anthropophilic, produced the mildest injection. The clinical stages of reiqfection were condensed for all three species, and all infected animals manifested delayed-type cutaneous h~persensitivit?: to homologous antigen, which persisted up to 12 months. There was a signi’cant MM1 und LT by cells from iqfected animuls exposed to homologous antigen. Cutaneous hypersensitivity ws transferred by splenic, lymph node, and peritoneal exudate cells from infected donors. The severity of primary injection correluted with the duration und magnitude of’reinfection symptoms. and with the persistence of h?persensitivity. These results indicate that there are systemic CMI responses to these fungi do not invade beyond the keratini:ed layers of skin, hair, and nails. rzhich 1rs14c411~~
The der matophytes, which
include species of and Epidermophyton, cause a variety of keratinized tissue infections. Many aspects of the host-parasite relationship remain obscure, including the role of immunological responses in these diseases. There is a large volume of literature on the immunology of dermatophytes, but available experimental and clinical data have not provided a model useful for control of the disease, or for explaining the many clinical variations of reinfection in humans. The question of systemic immune responses to these pathogens also is of interest because the determatophyte:; characteristically do not penetrate the epidermis. The foundations for studying cell-mediated immunity (CM111in response to dermatophytoses were laid in 1902, when Neisser’ reported sensitivity to “trichophytin. ” Many investigators have reported de-
Trichophyton,
layed-type hypersensitivity reactions to cellular filtrates or extracts of these fungi in humans and experimental animals (reviewed by Grappel, Bishop, and Blank*). Cruickshank, Trotter, and Wood3 were able to transfer delayed hypersensitivity (DHS) to trichophytin via peritoneal exudates, and more recently Jones, Reinhardt, and Rinaldi,” observed conversions to skin test antigen following controlled experimental infection of humans, and altered clinical course upon reinfection. The DHS reaction is not necessarily associated with acquired resistance, and the extensive reports establishing skin test reaction to trichophytin have not been matched with consistent data on acquired resistance to these fungi. In 1909 Bloch and Massini” reported that infections with these fungi produced a partial systemic immunity, but studies since then have failed to confirm this. Several workers found only a transient, localized resistance, and others have concluded that there is no acquired immune resistance to the dermatophytes in humans.” Jones, Reinhardt, and Rinaldi’ found that human subjects deficient in CM1 responses were susceptible to infection, but CM1 competence in that study was defined by sensitivity to the skin test antigen. These unresolved problems of the normal immune
Microsporum,
From the Institute of Postgraduate Medical Education and Research, Chandigarh, and the Department of Biology. University of Louisville. Received for publication April 11, 1978. Accepted for publication Nov. 21, 1978. Reprint reqwsts to: Dr. Lois S. Cronholm, Department of Biology, Universit) of Louisville, Louisville, KY 40208.
0091~67491’79/050361+09$00.9010
0 1979 The C. V. Mosby
Co.
Vol. 63, No. 5, pp. 361-369
362
Hunjan
and Cronholm
J. ALLERGY
i 10 Proie1n
5
lpg
protein
2.5 Concentration 101
1.25
0.62
0.15
ml1
FIG. 1. Magnitude of cutaneous hypersensitivity reactions with T. rubrum antigen. 0, Erythema; q , induration. Values are averages of 3 reactions measured 24 hr after intradermal injection of sensitized guinea pigs.
response to the dermatophytes point to the need for evidence on the systemic and local immune responses. The current study was designed as a systematic exploration of these immune responses in experimental animals using the major parameters of CMI: cutaneous delayed hypersensitivity; lymphocyte transformation (LT); macrophage migration inhibition (MMI); and passive transfer of hypersensitivity by immunocompetent cells. In addition, the alteration of clinical disease on reinfection was studied as an indirect index of a cell-mediated acquired immune response. MATERIALS
AND METHODS
T. rubrum, M. vanbreuseghemii, and E. joccosum were selected to represent the three genera of dermatophytes. T. rubrum and E. $occosum were isolated from patients treated at the Postgraduate Medical Institute Hospital, Chandigarh. M. vanbreuseghemii was supplied by Professor L. N. Mohapatra, Al 1 India Institute of Medical Sciences, New Delhi. The cultures were maintained on Difco Sabouraud’s dextrose-modified medium with chloramphenicol (0.05 mg/ml) and cyclohexamide (0.5 mg/ml). The final pH was 5.4 to 5.6. Cultures were incubated at 27” C. The test animals were guinea pigs, Hartley strain, 400 to 600 gm, of both sexes. The animals were fed water, a commercial balanced diet, green vegetables, and germinating Grams (Cicer arierimun L.) ad libitum. Each animal was caged separately
Preparation
and standardization
of antigens
The preparation of test antigen is a particularly critical facet of this study, since many of the uncertainties of responses to the dermatophytes are associated with problems of antigen preparation. In extensive pilot studies we prepared extracts by the most common methods reported for and tested each on guinea pigs reactive “trichophytin,”
CLIN. IMMUNOL. MAY 1979
sensitized by infection. This report includes only the details of that preparation which elicited the best cutaneous reactivity and which did not also act as a sensitizing agent. The antigens used in the study were prepared by ultrasonification of a saline homogenate of each species after growth for 3 to 4 wk at 27” C on the modified Sabouraud’s medium. The mycelial mat was washed three times in physiological saline and dried on filter paper. The dried preparation was ground with small aliquots of physiological saline, mixed with sterile glass powder in a sterile mortar, followed by further mixing in an M.S.E. Mullard homogenizer. These partially disintegrated cells were then subjected to sonification in an M.S.E. ultrasonic disintegrator at maximum kcisec for 15 min. The sonicated material was stored at 4” C for 24 to 48 hr, after which it was centrifuged at 2,000 to 3,000 rpm for IO to 15 min at 4” C. The supemate was sterilized by filtration, and each preparation was tested for sterility and dispensed in 5-ml aliquots, lyophilized, and stored at 4” C. The lyophilized antigen was reconstituted in distilled water for use. The dose of each antigen used was standardized according to protein content and in vivo reactivity in animals sensitized by infection. Protein determinations were made by the standard Lowry’s method, and verified by the micro-Kjeldahl’s nitrogen analysis described by Kabat and Mayer.’ The in vivo standards were based on cutaneous reactivity of sensitized animals. Five animals which had completed 4 infection cycles (techniques for infection are detailed below) were injected intradennally in the abdominal wall with 0.1 ml solutions containing antigen protein ranging from 0.15 pg to 40.0 pg (corresponding to dilutions ranging from neat to 1: 5 12). Control sensitized animals were injected with physiological saline. The skin reactions were observed at 15 min, 30 min, and 6 hr after treatment, and thereafter at 24-hr intervals for 14 days. Edema, erythema, and induration were the indices of reactivity, and were quantified by measurements of the inflamed site. The minimum dose of antigen protein which elicited a maximal response was used for all further studies; as shown in Fig. 1, this dose was 10.0 @g protein/O. 1 ml for T. rubrum. Corresponding data for the other 2 fungi led to doses of 1I .5 pg/O. 1 ml for M. vanbreuseghemii, and I1 .O pg/O. 1 ml for E. ~Yoccosu~, obtained in all cases by a 1: 4 dilution of the preparation.
Infection
and sensitization
of animals
Animals were inoculated with the three test fungi to produce symptoms suitable for descriptions of the primary infection and reinfection, and to sensitize the animals for assays of the immune response. The animals were prepared for primary infection by shaving a 10 cm2 area on one flank and scarifying 1 cm* of this area with a sterile blunt scalpel. Extreme care was taken to avoid traumatic inflammation. An 0.2 ml thick fungal slurry of a 3-wk culture of each fungus was prepared by suspension of the conidia and mycelia in physiological saline. This inoculum was gently but thoroughly rubbed into the scarified site. The same techniques were used for reinfection, except that previously
VOLUME NUMBER
Cell-mediated immune responses to dermatophytes
63 5
TABLE 1. The clinical
course of primary
363
disease Dermatophyte T. rubrum
Symptoms
M. vanbreuseghemii
1-3 3-5 5-16
4-5 6-11 12-16
Incubation period (days) Spreading of lesions (days) Climax of lesions (days) Positive skin scrapings* (days) Lesions healed (days) Characteristics of lesions
E. floccosum
4-5 6-12 13-14 23-24 34-36
24-27 45-50
20-27 30-35
Erythema I + Inflammation 3 + Scaling 3 +
Erythema 1+ Inflammation 4f Scaling I +
Erythema I+ Inflammation 1+ Scaling l+
*Skin scrapmgs from lesions were positive microscopically or culturally. TABLE II. The clinical
course of reinfection
disease Dermatophyte
T. rubrum Symptoms
(Numbers oi animals) Incubation period (days) Spreading of lesions (days) Climax of lesions (days) Positive skin scrapings (days) Lesions healed (days) Characteristics of lesions Erythema Inflammal:ion Scaling
M. vanbreuseghemii
E. floccosum
Sec.*
Tett.t
Quart.*
Sec.
Tert.
Quart.
Sec.
Tert.
Quart.
14 1-4
12 1-3
10 l-3
20
1-2
5-l 6-8 7-8
4-7 7-8 4-5 15-17
4-7 8-9 5-6 18-19
2-3 4-8 15-16 22-25
14 1-2 1-2 4-7 8-9 20-24
14 1-2 2-3 3-7 7-8 19-21
18 2-4 5-9 10-13 18-19 24-27
14 3-4 5-6 7-13 16-17 22-24
12 3-5 6-7 8-15 16-18 23-25
2+ 4+ 4+
2+ 4f 4+
1+
1+
1-t
4+ 3+
4+ 3+
4f 3+
1+ 1+
1+ 1+
1+ 1+
2+
2+
2+
12-15 2+ 4+ 4+
*Secondary i’lfection. tTertiary infection. $Quarternary infection. The animals were reinfected after healing of the lesions of the prior infection.
untreated pcttions of flank were inoculated. Reinfection inoculations were made 1 wk after the original lesions healed. Randomly selected groups of animals were subjected to secondary, lertiary, and quarternary reinfection. A total of 72 animals was studied for primary infection (24 for each of the 3 fungi), and a minimum of 10 animals was used for reinfection :studies. Randomly selected groups of animals studied for clinical responses were used for the studies of CMI.
Analysis infection
of clinical symptoms and reinfection
in primary
The clinical course of the primary and reinfection syndromes was measured by: incubation period; period of spreading to climax: duration of climax; duration of positive skin scrapings (fungi observed either microscopically or by culturing); the time to complete healing; characteristics of climax lesions (erythema. edema, and scaling). The reactions were scored on a scale from 1+ to 6+. All judgments of these reactions were made by the same invesiigator.
Analysis of the cell-mediated response: In vivo
immune
The development of cutaneous hypersensitivity during active infection was studied by inoculation of homologous antigen into animals during primary, secondary, tertiary, and quarternary infection. Control groups of uninfected animals given parallel inoculations indicated that the test antigen did not sensitize guinea pigs. The antigen extract of each fungus was injected intradermally into the abdomen of animals actively infected with that fungus. A fresh site was selected for each injection. The injections contained 10 pg to 11.5 pg antigen protein in a 0.1 ml inoculum administered every third day during active infection, and thereafter at weekly intervals until the animals became unresponsive to the antigen. Observations for delayed hypersensitivity were made every 24 hr after antigen challenge. The delayed-type reaction was measured with respect to erythema. edema, and induration. (Since eyrthema and edema are overlapping symptoms for infection and hypersensitivity, induration was the ma,jor criterion
364
Hunjan and Cronholm
J. ALLERGY
.-: 0
(months)
(days)
Time
FIG. 2. Intensity and duration of delayed-type hypersensitivity to T. rubrum antigen. Animals were challenged with T. rubrum antigen at 3-day intervals beginning with primary T. rubrum infection and continuing until animals became unresponsive.
.-ti 60 5 2 A5E l- / o 0-e 0
I
0
/ e-01 0-O % / ,,,;,\ I
a
I
1
l
16 24 32 Lo 48
(days)
246 (mom hs)
Time
FIG. 3. Intensity and duration of delayed-type hypersensitivity to E. floccosum antigen. Animals were challenged with E. floccosum antigen at 3-day intervals beginning with primary E. floccosum infection and continuing until animals became unresponsive.
for the hypersensitivity reaction.) Observations for immediate-type cutaneous reactions were also made, but these data are not presented in this paper.
Analysis of the cell-mediated response: In vitro
immune
The MM1 assay and LT assay for CM1 responses were performed on guinea pigs following quarternary infection, and on nonsensitized control guinea pigs. All infected animals exhibited cutaneous hypersensitivity responses to homologous test antigen and all uninfected controls gave negative skin reactions. The MM1 assay was performed by the methods of David and co-workers.g Peritoneal exudate cells were harvested from the guinea pigs following intraperitoneal injection of sterile light liquid paraffin. The capillary tubes packed with purified cell suspension were incubated in migration chambers with the purified antigen of each fungus in concentra-
CLIN. IMMUNOL. MAY 1979
tions of 50 pg protein/O. 1 ml for T. rcthrurn antigen, and 75 pg protein/O. 1 ml for M. ~dnw4seghernii and E. POE cosum antigen. The areas of migration were measured for macrophages from sensitized animals and nonsensitized animals after the cells were incubated with antigen, and for macrophages from sensitized animals after the cells were incubated in medium without antigen. The ratios of these migration areas were the indices of inhibition by antigen. For the LT assays, venous blood was obtained via cardiac puncture and 30 ml of blood was aspirated into sterile syringes with 1,500 U heparin and 2.0 ml of 6% dextran (average mw, 350,000). The blood was sedimented for 45 min at room temperature, and the leukocyte-rich plasma was recovered. Lymphocytes were isolated by centrifugation at 450 x g for 40 min at 18” C in a Ficoll-Hypaque gradient as described by Jondal, Holm, and Wigzell.“’ The lymphocytes were washed twice by centrifugation at 250 x g followed by 10 min centrifugation in TC-199 medium. The purified lymphocytes were resuspended to 1 x 10fi cells/ml of TC- 199 medium containing 20% heatinactivated guinea pig serum. These lymphocyte suspensions were dispensed as 1 ml aliquots in test tubes to which 0.1 ml of the antigen or control substance was added. Controls included: TC- 199 medium, phytohemagglutinin (PHA), and streptokinase-streptodomase (SK-SD). Nonstimulated control assays (TC-199 medium) were performed in quadruplicate. Stimulated control and experimental assays (PHA, SK-SD, and test antigen) were performed in triplicate. Cultures were incubated for 5 days at 37” C in a 5% CO* humidified atmosphere, and the experimental and control mitogens were added at day 2. Five hr prior to harvest, 1.O &i of tritiated thymidine (AmershamiSearle. Arlington Heights, Ill.), specific activity 17 Ci/mmol, was added to each preparation. The cells were harvested by trichloroacetic acid precipitation as described by Valentine.” The precipitates were solubilized in 1.0 ml NCS (Amersham/Searle) and then quantitatively transferred to scintillation vials with 9.0 ml of a toluene-based scintillation fluid. Radioactivity was measured with a Beckman LS- 150 Liquid scintillation counter. The blastogenic index (BI) was calculated as the ratio of the counts per minute of antigen-stimulated culture to the control (TC- 199 medium) cultures.
Passive transfer
of delayed
hypersensitivity
The sensitized animals used as sources of macrophage for the MM1 were also used as donors for assays of passive transfer of hypersensitivity to the antigen. The methods of Chase’* and Haxthusen’” were used essentially without modification. The donor cells included populations derived from the spleen, inguinal lymph nodes, peritoneal exudates, and lymphocytes obtained via cardiac puncture. Viability of the donor cells was determined by the Trypan blue dye exclusion test’” and transfers were made within 1 hr of the test. The cells were inoculated intravenously into clinically healthy recipient female guinea pigs in quantities shown in the section on results. The ability of the donor cells to confer hypersensitivity to the recipient was deter-
VOLUME NUMBER
Cell-mediated
63 5
immune
responses
I
(days1
Time
(months)
FIG. 4. Inter&y and duration of delayed-type hypersensitivity to hl. vanbreuseghemii antigen. Animals were challenged with M. vanbreuseghemii antigen at 3-day intervals beginning with primary M. vanbreuseghemii infection and continuing until animals became unresponsive.
mined by reaction to intradermal injection of homologous test antigen. The test reactions were recorded from 24 hr to 15 days after challenge.
RESULTS Clinical symptoms of primary reinfection disease
and
The symptoms observed in primary disease of guinea pigs inoculated with the three dermatophytes are shown in Table I. The disease produced by M. vanbreuseg.hemii was considered the most virulent, based on incubation period, time to healing, and degree of inflammation, even though the period of positive skin scraping was similar to the other two fungi. The clinical course elicited by T. rubrum and E. jloccosum was similar with respect to the time from incubation period to healing, but the degree of inflammation was more severe for T. rubrum. The relative severity of these three diseases was therefore scored as M. \!anbreuseghemii > T. rubrum > E. jotcosum
The clinical symptoms of reinfection are shown in Table II. The duration of the clinical symptoms from incubation period to healing was shortened in all cases of reinfection, although the differences between duration of stages of primary and secondary infection were greater than the differences between secondary, tertiary, and quarternary infection. There was an apparent correlation between virulence of primary disease and altered duration of the reinfection symptoms. This same relationship held in the magnitude of the response du.ring the maximum period of inflammation in that E. joccosum failed to elicit severe reactions, while the responses to T. rubrum and M. van-
to dermatophytes
Ill II Groups
365
IV
FIG. 5. Inhibition of macrophage migration. Groups I, II, and Ill had been infected with T. rubrum (I), M. vanbreuseghemii (II), or E. floccosum (Ill), and all exhibited cutaneous hypersensitivity responses to homologous test antigen. Group IV animals were uninfected and gave negative skin reactions to test antigens. Macrophage populations from each group were incubated with test antigen or control medium following quarternary infection (for I, II, and III): UPJ,T. rubrum antigen; 5, M. vanbreuseghemii antigen; 8. E. floccosum antigen; 0, control medium.
:
60
G ro ups FIG. 6. Lymphocyte transformation. Group I animals had completed quarternary infections with T. rubrum, Group II with M. vanbreuseghemii, and Group Ill with E. floccosum, and all exhibited cutaneous hypersensitivity to homologous antigen. Values on left are responses of lymphocytes incubated with homologous antigen; values on right are responses of lymphocytes incubated with control medium.
were enhanced by re-exposure. The increased severity of these two in reinfection was manifest partly by extensive shedding of the comeum stratum, occasionally leading to the formation of crusts followed by sloughing. The relationship between the period of positive skin scrapings and the length of time to healing was also altered, particularly in the tertiary and quartemary infections of T. rubrum and M. vanbreuseghemii. For example, in primary M.
breuseghemii
366 Hunjan and Cronholm
TABLE
III. Delayed
J. ALLERGY
hypersensitivity
reactions
accompanying
dermatophyte
CLIN. IMMUNOL. MAY 1979
infection
Dermatophyte Infection
cycle
Primary
Secondary
Tertiary
Quarternary
Course of hypersensitivity
T. rubrum
Onset (days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days)
7 49 to 56 4.0 to 6.5 10 to 14
10 46 3.5 to 5.5 9to 11
10 50 3.5 to 5.0 10 to 12
NR*
8 4.5 to 6.5 9to 15
12 4.0 to 6.0 11 to 15
16 5.0 to 5.5 11 to 13
NR
7 5.0 to 6.5 11 to 16
12 4.0 to 6.0 11 to 18
12 5.0 to 5.5 10 to 14
NR
11 5.5 to 6.5 10 to 17
12 4.0 to 6.0 11 to 17
12 5.0 to 5.5 12 to 15
NR
*NR, nonreactive (controls inoculated with physiological
infection, the fungus was demonstrable for 24 to 27 days, and the lesions healed by the fiftieth day; in the quarternary infection, the fungus was not found after 8 days, but the lesions did not heal for 19 to 21 days. The shortened period of positive skin scrapings is explained by an immune response, and the prolonged period to healing suggests an overlapping of symptoms of infection and hypersensitivity. Histological preparation of the skin lesions verified that the fungi did not penetrate below the comeum stratum, and lay parallel to the epidermal layers. hypersensitivity:
E. floccosum
Controls
saline).
vanbreuseghemii
Delayed
M. vanbreuseghemii
In vivo responses
The results of intradermal challenge of infected animals with test antigens are shown in Table III and Figs. 2 to 4. The onset of a delayed hypersensitivity response coincided with the developmental or early climax stage of the lesions. The maximum response during primary infection was reached at approximately the same time for all three fungi, even though the clinical course varied. These marked differences in infection and reinfection were also absent in the magnitude of the hypersensitivity response to the three fungi. The time to maximal hypersensitivity response in reinfection was considerably shorter than in primary infection, and there was a moderate increase in the size of the indurated area during reinfection.
The persistance of the delayed cutaneous reaction is shown in Figs. 2 to 4. These data show the responses during active infection and after healing of the final experimental infection until the animals became nonreactive. The hypersensitivity diminished over a period of twelve months for T. rubrum and ten months for M. vanbreuseghemii, but it diminished abruptly for E. jloccosum and was absent by six months. The skin test antigen did not interfere with the DHS or with clinical infection. Control uninfected animals inoculated with the three test antigens in parallel with the infected animals did not develop cutaneous DHS responses, and these animals were susceptible to infection when they were inoculated with the fungi after this prolonged exposure to the test antigen. Delayed
hypersensitivity:
In vitro
responses
The results of the migration inhibition assay are shown graphically in Fig. 5. There was a significant difference between the MM1 of controls and the experimental groups (p < 0.05, Student’s t test), and inhibition reached 60% in the case of M. vanbreuseghetnii. The percentage inhibition by T. rubrum antigen was smaller than the response to the other two fungal antigens, but the difference was not significant (p < 0.05, Student’s t test). The results of the LT assays for specific antigenic
VOLUME NUMBER
TABLE
Cell-mediated immune responses to dermatophytes
63 5
IV. Passive
transfer
of cutaneous
sensitivity
in guinea
lntradermal Recipient’ TM Et
ii 2$ 3i: 4$ 5$ 63 I 8 9 10 II c c c c
12 c c
13 14 16 17 c c c c ccc
15 18 c c
19$20$2 I 22$23 241 25 26 21 28 29$30 ccc ccc
31432 334 34335 36; 37 38$39 40 41$42 c c c c c c
Number of ceils transferred
3.0-5.0 x IOR
1.0 x 10”
2.0-2.5 x 108
2.0-2.5 x 108
24 hr TME
46 hr TME
Splenic cells +-+ +-+++ +--
Peripheral +++ +----
9 days
12days
15days
TME
TME
TME
t-+ +++ -+-
f-+ +++ -+-
f-f +++ -+---
+-+++ -+-
-+-
leukocytes +++ +++ +-+-+++ +++ -----
+--
test reaction
6 days TME
Peritoneal exudate c~ells +++ +++ +-++++++-
pigs
72 hr TME
Inguinal lymph node cells +-+-+------+-
++-
367
Positive transfers
T
M
E
2/4 2/4 2/4
---
1/2 l/2 o/2
+++ +--++
-++ +--
3/4 2/4 2/4
+++ ++-
+++ ++-
-++--
++-
-+-
---
+--+-
-+-
-t-t+ +-+++
3/4 3/4 l/4 +--
C, Controls receiving cells from nonsensitized donors. *Animals 1-42 were 2-3 wk old guinea pigs receiving cells from sensitized donors. tT, T. ruhrum; M, M. vanhreuseghemii; E, E. floccosum $The progeny of the donor.
activators are depicted in Fig. 6. In the controls with nonspecific mitogens, the 3H-thymidine uptake of the 1ymphocyl:es of sensitized and nonsensitized animals indicated :significant activation. The response of sensitized animals was not significantly higher than that of the nonsensitized, although it is interesting that lymphocytes from the recently infected animals tended to be more responsive. There was a significant difference in the response of the same lymphocyte populations from the experimental and control groups to the specific antigenic activators. As in the macrophage inhibition assay, the response to T. rubrum antigen was less than the other two antigens, but there was no significant difference between the responses in the three experimental groups (p < 0.05, Student’s t test).
Passive transfer
of sensitivity
The results of the studies on passive transfer of delayed hypersensitivity to the dermatophytes are shown in Table IV. Donor splenic cells and peripheral leukocytes sensitized 50% to 75% of all the recipients. Peritoneal exudate cells sensitized some animals in each group, but only 1 of 4 recipients became responsive to the E. jbxosum antigen. The only donor populations which failed to elicit a response were cells from the inguinal lymph nodes in the case of E. poccosum; however, the lymph node cells sensitized 50% of the animals tested with the other two fungi. The passively transferred cutaneous sensitivity was short-lived and only two animals remained sensitized on the last day of the experiment (fifteenth day).
366
Hunjan and Cronholm
DISCUSSION Infection by dermatophytes undoubtedly may be accompanied by an acquired immune response. This is verified primarily by the extensive documentation that cutaneous sensitivity is a common correlate to infection. The major unresolved issues include the relationship between this sensitization and a predictable alteration in the status of the host to reinfection. This information is essential for an understanding of the basic mechanisms of these diseases, and to any rational program of disease management by immunological methods. These issues are also of theoretical interest because they include the interesting problem of systemic responses to noninvasive pathogens. A single assay for hypersensitivity is inconclusive evidence that the full range of CM1 responses is operative. The direct evidence for CM1 to dermatophytes is found primarily in the reports of local hypersensitivity reactions. This study combined in vitro and in vivo assays for systemic CM1 as correlates to these local reactions. The results of this systematic investigation present compelling evidence that a full array of CM1 is associated with these fungal infections. Clinically, the course of the disease was altered in a pattern suggesting acquired immunity, with a marked condensation of the infectious phases and variations in the lesions, which are reasonably explained as an exacerbation by the inflammatory stages of cutaneous hypersensitivity. This shift in the clinical course upon reinfection of animals was also reported by DeLamater and Benham15 and in humans by Jones, Reinhardt, and Rinaldi .4 The specific course of alteration in reinfection for the three dermatophytes bore an interesting relationship to the primary disease, and verifies the correspondence noted by others between immunity and host range.2 M. vanbreuseghemii, a zoophilic species, elicited a severe inflammatory response in primary disease and reinfection. T. rubrum, which is primarily anthropophilic but is known to infect other species, produced a moderate inflammatory response in the primary infection and a severe inflammation in reinfection. E. poccosum, previously considered obligately anthropophilic, produced only a mild inflammation in all infections. The course of the cutaneous hypersensitivity developing during infection was similar to other reports,’ and indicates that the immune status of our experimental animals was similar to equivalent systems, including humans. There was consistency between the data obtained on persistence of cutaneous hypersensitivity after infection and the clinical pattern in reinfection. The pathogen which elicited the mildest
J. ALLERGY
CLIN. IMMUNOL. MAY 1979
symptoms on primary infection and the least alteration on reinfection also produced a short-lived hypersensitivity. Two major contributions of this study are the MM1 and LT assays, which are important confirmations of the sensitization of systemic immunocompetent cells to the antigens which yield positive skiI! tests. The inhibition of macrophage migration and activation of the lymphocytes by specific antigen should be interpreted as direct evidence for a systemic response to these fungi. The results of the passive tracsfer by cells is less satisfying because syngeneic animals were unavailable, and because the populations of donor cells were mixed. However, these data present valid additive evidence of a systemic CM1 response. The relative magnitude of infection and the LT and MM1 responses were not as consistent as the reinfection and cutaneous hypersensitivity reactions, in that E. jotcosum antigen and E.$occosum-sensitized cells were more reactive than the T. rubrum systems. However, the correspondence with clinical syndrome (and host range) was observed in the relative effectiveness of passive transfer of hypersensitivity by M. vanbreuseghemii- and E. jfoccosum- sensitized cells. This study does not resolve the inconsistencies between the experimental and clinical data on human hosts, which show that the altered immunity found in experimental conditions is not reflected in resistance to natural reinfection. It must be emphasized that such CM1 responses as DHS do not necessarily correlate with resistance. Further, many factors in natural infection, including variations in host immune competence and exposure to cross-reacting antigens, may alter the individual clinical pattern. There might also be differences in the distribution of diffusible antigen as a function of the specific host-parasite relationship. Such variations must be considered within a general model for a CM1 response to dermatophytes that is confirmed by this study. REFERENCES 1. Neisser A: Plato’s versuche uber die Herstellung und Verwendung von “Trichophytin,” Arch Dermatol60:63, 1902. 2. Grappel SF, Bishop CT, Blank F: Immunology of dermatophytes and dermatophytosis, Bacterial Rev 38:222, 1974. 3. Cruickshank CND, Trotter MD, Wood SO: Studies on trichophytin sensitivity, .I Invest Dermatol 35219, 1960. 4. Jones HE, Reinhardt JH, Rinaldi MG: Acquired immunity to dermatophytes, Arch Dermatol 109:840, 1974. 5. Bloch B, Massini R: Studien uber Immunitat und Uberempfindichkeit bei Hyphomycetener Krankungen, Z Hyg Infeksionskr 6368, 1909. 6. Sloper JC: A study of experimental human infections due to Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton j7occosum, with particular reference to the
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Cell-mediated
63 5
self-limitation of the resultant lesions, J Invest Dermatol 25:21, 1955. Jones HE. Reinhardt JH, Rinaldi MG: Model dermatophytosis in naturally infected subjects, Arch Dermatol 110~369, 1974. Kabat EA. Mayer MD: Experimental immunochemistry, Springfield, Ill., 1948, Charles C Thomas, Publishers. David JR, Al-Askari S, Lawrence HS, Thomas L: Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigen, J. lmmunol 93~264, 1964. Jondal M, Holm G, Wigzell H: Surface markers of T and B lymphocytes, J Exp Med 136~207, 1972. Valentine F: Lymphocyte transformation, in Bloom BR, Glade
Information
12. 13. 14. 15.
immune
responses
to dermatophytes
369
PP. editors:/n vim methods in cell mediated immunity, 197 1, Academic Press Inc., New York. Chase M: The cellular transfer of cutaneous hypersensitivity. J Bacterial 51:643, 1946. Haxthausen H: Experiments on passive transfer of eczematous allergy, J Invest Dermatol 19:293, 1952. Howard DH: Fate of Hisroplasma capsulutum in guinea pig polymorphonuclear leucocytes, Infect Immun 8~412, 1973. DeLamater ED, Benham RW: Experimental studies with the dermatophytes. II. Immunity and hypersensitivity produced in laboratory animals, J Invest Dermatol 1:469, 1938.
for authors
Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership of the manuscript (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. 1 sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.
Ph.D., and L. S. Cronholm,
Ph.D. Chandigarh, India,
and
KY.
Guinea pigs in&ted with three species of dermatophytes were tested for cell-mediated immune responses, incYuding delayed cutuneous hypersen.~itiL’ity. (w~phocyte transf&mation (LT), muc,rophuge migration inhibition (MMIJ. and passive tran?fer of hypersensitivity. The patterns of reinfection disease were also unalyzed as an index of altered host immunity. The fungi tested represented the three genera oj’derrnatoph~tes (Trichophyton, Microsporum, and Epidennophyton), and included zoophilic and anthropophilic, species. Microsporum vanbreuseghemii, the zoophilic species, produced the most virulent infection, and Epidemwphyton floccosum, previously considered obligutely anthropophilic, produced the mildest injection. The clinical stages of reiqfection were condensed for all three species, and all infected animals manifested delayed-type cutaneous h~persensitivit?: to homologous antigen, which persisted up to 12 months. There was a signi’cant MM1 und LT by cells from iqfected animuls exposed to homologous antigen. Cutaneous hypersensitivity ws transferred by splenic, lymph node, and peritoneal exudate cells from infected donors. The severity of primary injection correluted with the duration und magnitude of’reinfection symptoms. and with the persistence of h?persensitivity. These results indicate that there are systemic CMI responses to these fungi do not invade beyond the keratini:ed layers of skin, hair, and nails. rzhich 1rs14c411~~
The der matophytes, which
include species of and Epidermophyton, cause a variety of keratinized tissue infections. Many aspects of the host-parasite relationship remain obscure, including the role of immunological responses in these diseases. There is a large volume of literature on the immunology of dermatophytes, but available experimental and clinical data have not provided a model useful for control of the disease, or for explaining the many clinical variations of reinfection in humans. The question of systemic immune responses to these pathogens also is of interest because the determatophyte:; characteristically do not penetrate the epidermis. The foundations for studying cell-mediated immunity (CM111in response to dermatophytoses were laid in 1902, when Neisser’ reported sensitivity to “trichophytin. ” Many investigators have reported de-
Trichophyton,
layed-type hypersensitivity reactions to cellular filtrates or extracts of these fungi in humans and experimental animals (reviewed by Grappel, Bishop, and Blank*). Cruickshank, Trotter, and Wood3 were able to transfer delayed hypersensitivity (DHS) to trichophytin via peritoneal exudates, and more recently Jones, Reinhardt, and Rinaldi,” observed conversions to skin test antigen following controlled experimental infection of humans, and altered clinical course upon reinfection. The DHS reaction is not necessarily associated with acquired resistance, and the extensive reports establishing skin test reaction to trichophytin have not been matched with consistent data on acquired resistance to these fungi. In 1909 Bloch and Massini” reported that infections with these fungi produced a partial systemic immunity, but studies since then have failed to confirm this. Several workers found only a transient, localized resistance, and others have concluded that there is no acquired immune resistance to the dermatophytes in humans.” Jones, Reinhardt, and Rinaldi’ found that human subjects deficient in CM1 responses were susceptible to infection, but CM1 competence in that study was defined by sensitivity to the skin test antigen. These unresolved problems of the normal immune
Microsporum,
From the Institute of Postgraduate Medical Education and Research, Chandigarh, and the Department of Biology. University of Louisville. Received for publication April 11, 1978. Accepted for publication Nov. 21, 1978. Reprint reqwsts to: Dr. Lois S. Cronholm, Department of Biology, Universit) of Louisville, Louisville, KY 40208.
0091~67491’79/050361+09$00.9010
0 1979 The C. V. Mosby
Co.
Vol. 63, No. 5, pp. 361-369
362
Hunjan
and Cronholm
J. ALLERGY
i 10 Proie1n
5
lpg
protein
2.5 Concentration 101
1.25
0.62
0.15
ml1
FIG. 1. Magnitude of cutaneous hypersensitivity reactions with T. rubrum antigen. 0, Erythema; q , induration. Values are averages of 3 reactions measured 24 hr after intradermal injection of sensitized guinea pigs.
response to the dermatophytes point to the need for evidence on the systemic and local immune responses. The current study was designed as a systematic exploration of these immune responses in experimental animals using the major parameters of CMI: cutaneous delayed hypersensitivity; lymphocyte transformation (LT); macrophage migration inhibition (MMI); and passive transfer of hypersensitivity by immunocompetent cells. In addition, the alteration of clinical disease on reinfection was studied as an indirect index of a cell-mediated acquired immune response. MATERIALS
AND METHODS
T. rubrum, M. vanbreuseghemii, and E. joccosum were selected to represent the three genera of dermatophytes. T. rubrum and E. $occosum were isolated from patients treated at the Postgraduate Medical Institute Hospital, Chandigarh. M. vanbreuseghemii was supplied by Professor L. N. Mohapatra, Al 1 India Institute of Medical Sciences, New Delhi. The cultures were maintained on Difco Sabouraud’s dextrose-modified medium with chloramphenicol (0.05 mg/ml) and cyclohexamide (0.5 mg/ml). The final pH was 5.4 to 5.6. Cultures were incubated at 27” C. The test animals were guinea pigs, Hartley strain, 400 to 600 gm, of both sexes. The animals were fed water, a commercial balanced diet, green vegetables, and germinating Grams (Cicer arierimun L.) ad libitum. Each animal was caged separately
Preparation
and standardization
of antigens
The preparation of test antigen is a particularly critical facet of this study, since many of the uncertainties of responses to the dermatophytes are associated with problems of antigen preparation. In extensive pilot studies we prepared extracts by the most common methods reported for and tested each on guinea pigs reactive “trichophytin,”
CLIN. IMMUNOL. MAY 1979
sensitized by infection. This report includes only the details of that preparation which elicited the best cutaneous reactivity and which did not also act as a sensitizing agent. The antigens used in the study were prepared by ultrasonification of a saline homogenate of each species after growth for 3 to 4 wk at 27” C on the modified Sabouraud’s medium. The mycelial mat was washed three times in physiological saline and dried on filter paper. The dried preparation was ground with small aliquots of physiological saline, mixed with sterile glass powder in a sterile mortar, followed by further mixing in an M.S.E. Mullard homogenizer. These partially disintegrated cells were then subjected to sonification in an M.S.E. ultrasonic disintegrator at maximum kcisec for 15 min. The sonicated material was stored at 4” C for 24 to 48 hr, after which it was centrifuged at 2,000 to 3,000 rpm for IO to 15 min at 4” C. The supemate was sterilized by filtration, and each preparation was tested for sterility and dispensed in 5-ml aliquots, lyophilized, and stored at 4” C. The lyophilized antigen was reconstituted in distilled water for use. The dose of each antigen used was standardized according to protein content and in vivo reactivity in animals sensitized by infection. Protein determinations were made by the standard Lowry’s method, and verified by the micro-Kjeldahl’s nitrogen analysis described by Kabat and Mayer.’ The in vivo standards were based on cutaneous reactivity of sensitized animals. Five animals which had completed 4 infection cycles (techniques for infection are detailed below) were injected intradennally in the abdominal wall with 0.1 ml solutions containing antigen protein ranging from 0.15 pg to 40.0 pg (corresponding to dilutions ranging from neat to 1: 5 12). Control sensitized animals were injected with physiological saline. The skin reactions were observed at 15 min, 30 min, and 6 hr after treatment, and thereafter at 24-hr intervals for 14 days. Edema, erythema, and induration were the indices of reactivity, and were quantified by measurements of the inflamed site. The minimum dose of antigen protein which elicited a maximal response was used for all further studies; as shown in Fig. 1, this dose was 10.0 @g protein/O. 1 ml for T. rubrum. Corresponding data for the other 2 fungi led to doses of 1I .5 pg/O. 1 ml for M. vanbreuseghemii, and I1 .O pg/O. 1 ml for E. ~Yoccosu~, obtained in all cases by a 1: 4 dilution of the preparation.
Infection
and sensitization
of animals
Animals were inoculated with the three test fungi to produce symptoms suitable for descriptions of the primary infection and reinfection, and to sensitize the animals for assays of the immune response. The animals were prepared for primary infection by shaving a 10 cm2 area on one flank and scarifying 1 cm* of this area with a sterile blunt scalpel. Extreme care was taken to avoid traumatic inflammation. An 0.2 ml thick fungal slurry of a 3-wk culture of each fungus was prepared by suspension of the conidia and mycelia in physiological saline. This inoculum was gently but thoroughly rubbed into the scarified site. The same techniques were used for reinfection, except that previously
VOLUME NUMBER
Cell-mediated immune responses to dermatophytes
63 5
TABLE 1. The clinical
course of primary
363
disease Dermatophyte T. rubrum
Symptoms
M. vanbreuseghemii
1-3 3-5 5-16
4-5 6-11 12-16
Incubation period (days) Spreading of lesions (days) Climax of lesions (days) Positive skin scrapings* (days) Lesions healed (days) Characteristics of lesions
E. floccosum
4-5 6-12 13-14 23-24 34-36
24-27 45-50
20-27 30-35
Erythema I + Inflammation 3 + Scaling 3 +
Erythema 1+ Inflammation 4f Scaling I +
Erythema I+ Inflammation 1+ Scaling l+
*Skin scrapmgs from lesions were positive microscopically or culturally. TABLE II. The clinical
course of reinfection
disease Dermatophyte
T. rubrum Symptoms
(Numbers oi animals) Incubation period (days) Spreading of lesions (days) Climax of lesions (days) Positive skin scrapings (days) Lesions healed (days) Characteristics of lesions Erythema Inflammal:ion Scaling
M. vanbreuseghemii
E. floccosum
Sec.*
Tett.t
Quart.*
Sec.
Tert.
Quart.
Sec.
Tert.
Quart.
14 1-4
12 1-3
10 l-3
20
1-2
5-l 6-8 7-8
4-7 7-8 4-5 15-17
4-7 8-9 5-6 18-19
2-3 4-8 15-16 22-25
14 1-2 1-2 4-7 8-9 20-24
14 1-2 2-3 3-7 7-8 19-21
18 2-4 5-9 10-13 18-19 24-27
14 3-4 5-6 7-13 16-17 22-24
12 3-5 6-7 8-15 16-18 23-25
2+ 4+ 4+
2+ 4f 4+
1+
1+
1-t
4+ 3+
4+ 3+
4f 3+
1+ 1+
1+ 1+
1+ 1+
2+
2+
2+
12-15 2+ 4+ 4+
*Secondary i’lfection. tTertiary infection. $Quarternary infection. The animals were reinfected after healing of the lesions of the prior infection.
untreated pcttions of flank were inoculated. Reinfection inoculations were made 1 wk after the original lesions healed. Randomly selected groups of animals were subjected to secondary, lertiary, and quarternary reinfection. A total of 72 animals was studied for primary infection (24 for each of the 3 fungi), and a minimum of 10 animals was used for reinfection :studies. Randomly selected groups of animals studied for clinical responses were used for the studies of CMI.
Analysis infection
of clinical symptoms and reinfection
in primary
The clinical course of the primary and reinfection syndromes was measured by: incubation period; period of spreading to climax: duration of climax; duration of positive skin scrapings (fungi observed either microscopically or by culturing); the time to complete healing; characteristics of climax lesions (erythema. edema, and scaling). The reactions were scored on a scale from 1+ to 6+. All judgments of these reactions were made by the same invesiigator.
Analysis of the cell-mediated response: In vivo
immune
The development of cutaneous hypersensitivity during active infection was studied by inoculation of homologous antigen into animals during primary, secondary, tertiary, and quarternary infection. Control groups of uninfected animals given parallel inoculations indicated that the test antigen did not sensitize guinea pigs. The antigen extract of each fungus was injected intradermally into the abdomen of animals actively infected with that fungus. A fresh site was selected for each injection. The injections contained 10 pg to 11.5 pg antigen protein in a 0.1 ml inoculum administered every third day during active infection, and thereafter at weekly intervals until the animals became unresponsive to the antigen. Observations for delayed hypersensitivity were made every 24 hr after antigen challenge. The delayed-type reaction was measured with respect to erythema. edema, and induration. (Since eyrthema and edema are overlapping symptoms for infection and hypersensitivity, induration was the ma,jor criterion
364
Hunjan and Cronholm
J. ALLERGY
.-: 0
(months)
(days)
Time
FIG. 2. Intensity and duration of delayed-type hypersensitivity to T. rubrum antigen. Animals were challenged with T. rubrum antigen at 3-day intervals beginning with primary T. rubrum infection and continuing until animals became unresponsive.
.-ti 60 5 2 A5E l- / o 0-e 0
I
0
/ e-01 0-O % / ,,,;,\ I
a
I
1
l
16 24 32 Lo 48
(days)
246 (mom hs)
Time
FIG. 3. Intensity and duration of delayed-type hypersensitivity to E. floccosum antigen. Animals were challenged with E. floccosum antigen at 3-day intervals beginning with primary E. floccosum infection and continuing until animals became unresponsive.
for the hypersensitivity reaction.) Observations for immediate-type cutaneous reactions were also made, but these data are not presented in this paper.
Analysis of the cell-mediated response: In vitro
immune
The MM1 assay and LT assay for CM1 responses were performed on guinea pigs following quarternary infection, and on nonsensitized control guinea pigs. All infected animals exhibited cutaneous hypersensitivity responses to homologous test antigen and all uninfected controls gave negative skin reactions. The MM1 assay was performed by the methods of David and co-workers.g Peritoneal exudate cells were harvested from the guinea pigs following intraperitoneal injection of sterile light liquid paraffin. The capillary tubes packed with purified cell suspension were incubated in migration chambers with the purified antigen of each fungus in concentra-
CLIN. IMMUNOL. MAY 1979
tions of 50 pg protein/O. 1 ml for T. rcthrurn antigen, and 75 pg protein/O. 1 ml for M. ~dnw4seghernii and E. POE cosum antigen. The areas of migration were measured for macrophages from sensitized animals and nonsensitized animals after the cells were incubated with antigen, and for macrophages from sensitized animals after the cells were incubated in medium without antigen. The ratios of these migration areas were the indices of inhibition by antigen. For the LT assays, venous blood was obtained via cardiac puncture and 30 ml of blood was aspirated into sterile syringes with 1,500 U heparin and 2.0 ml of 6% dextran (average mw, 350,000). The blood was sedimented for 45 min at room temperature, and the leukocyte-rich plasma was recovered. Lymphocytes were isolated by centrifugation at 450 x g for 40 min at 18” C in a Ficoll-Hypaque gradient as described by Jondal, Holm, and Wigzell.“’ The lymphocytes were washed twice by centrifugation at 250 x g followed by 10 min centrifugation in TC-199 medium. The purified lymphocytes were resuspended to 1 x 10fi cells/ml of TC- 199 medium containing 20% heatinactivated guinea pig serum. These lymphocyte suspensions were dispensed as 1 ml aliquots in test tubes to which 0.1 ml of the antigen or control substance was added. Controls included: TC- 199 medium, phytohemagglutinin (PHA), and streptokinase-streptodomase (SK-SD). Nonstimulated control assays (TC-199 medium) were performed in quadruplicate. Stimulated control and experimental assays (PHA, SK-SD, and test antigen) were performed in triplicate. Cultures were incubated for 5 days at 37” C in a 5% CO* humidified atmosphere, and the experimental and control mitogens were added at day 2. Five hr prior to harvest, 1.O &i of tritiated thymidine (AmershamiSearle. Arlington Heights, Ill.), specific activity 17 Ci/mmol, was added to each preparation. The cells were harvested by trichloroacetic acid precipitation as described by Valentine.” The precipitates were solubilized in 1.0 ml NCS (Amersham/Searle) and then quantitatively transferred to scintillation vials with 9.0 ml of a toluene-based scintillation fluid. Radioactivity was measured with a Beckman LS- 150 Liquid scintillation counter. The blastogenic index (BI) was calculated as the ratio of the counts per minute of antigen-stimulated culture to the control (TC- 199 medium) cultures.
Passive transfer
of delayed
hypersensitivity
The sensitized animals used as sources of macrophage for the MM1 were also used as donors for assays of passive transfer of hypersensitivity to the antigen. The methods of Chase’* and Haxthusen’” were used essentially without modification. The donor cells included populations derived from the spleen, inguinal lymph nodes, peritoneal exudates, and lymphocytes obtained via cardiac puncture. Viability of the donor cells was determined by the Trypan blue dye exclusion test’” and transfers were made within 1 hr of the test. The cells were inoculated intravenously into clinically healthy recipient female guinea pigs in quantities shown in the section on results. The ability of the donor cells to confer hypersensitivity to the recipient was deter-
VOLUME NUMBER
Cell-mediated
63 5
immune
responses
I
(days1
Time
(months)
FIG. 4. Inter&y and duration of delayed-type hypersensitivity to hl. vanbreuseghemii antigen. Animals were challenged with M. vanbreuseghemii antigen at 3-day intervals beginning with primary M. vanbreuseghemii infection and continuing until animals became unresponsive.
mined by reaction to intradermal injection of homologous test antigen. The test reactions were recorded from 24 hr to 15 days after challenge.
RESULTS Clinical symptoms of primary reinfection disease
and
The symptoms observed in primary disease of guinea pigs inoculated with the three dermatophytes are shown in Table I. The disease produced by M. vanbreuseg.hemii was considered the most virulent, based on incubation period, time to healing, and degree of inflammation, even though the period of positive skin scraping was similar to the other two fungi. The clinical course elicited by T. rubrum and E. jloccosum was similar with respect to the time from incubation period to healing, but the degree of inflammation was more severe for T. rubrum. The relative severity of these three diseases was therefore scored as M. \!anbreuseghemii > T. rubrum > E. jotcosum
The clinical symptoms of reinfection are shown in Table II. The duration of the clinical symptoms from incubation period to healing was shortened in all cases of reinfection, although the differences between duration of stages of primary and secondary infection were greater than the differences between secondary, tertiary, and quarternary infection. There was an apparent correlation between virulence of primary disease and altered duration of the reinfection symptoms. This same relationship held in the magnitude of the response du.ring the maximum period of inflammation in that E. joccosum failed to elicit severe reactions, while the responses to T. rubrum and M. van-
to dermatophytes
Ill II Groups
365
IV
FIG. 5. Inhibition of macrophage migration. Groups I, II, and Ill had been infected with T. rubrum (I), M. vanbreuseghemii (II), or E. floccosum (Ill), and all exhibited cutaneous hypersensitivity responses to homologous test antigen. Group IV animals were uninfected and gave negative skin reactions to test antigens. Macrophage populations from each group were incubated with test antigen or control medium following quarternary infection (for I, II, and III): UPJ,T. rubrum antigen; 5, M. vanbreuseghemii antigen; 8. E. floccosum antigen; 0, control medium.
:
60
G ro ups FIG. 6. Lymphocyte transformation. Group I animals had completed quarternary infections with T. rubrum, Group II with M. vanbreuseghemii, and Group Ill with E. floccosum, and all exhibited cutaneous hypersensitivity to homologous antigen. Values on left are responses of lymphocytes incubated with homologous antigen; values on right are responses of lymphocytes incubated with control medium.
were enhanced by re-exposure. The increased severity of these two in reinfection was manifest partly by extensive shedding of the comeum stratum, occasionally leading to the formation of crusts followed by sloughing. The relationship between the period of positive skin scrapings and the length of time to healing was also altered, particularly in the tertiary and quartemary infections of T. rubrum and M. vanbreuseghemii. For example, in primary M.
breuseghemii
366 Hunjan and Cronholm
TABLE
III. Delayed
J. ALLERGY
hypersensitivity
reactions
accompanying
dermatophyte
CLIN. IMMUNOL. MAY 1979
infection
Dermatophyte Infection
cycle
Primary
Secondary
Tertiary
Quarternary
Course of hypersensitivity
T. rubrum
Onset (days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days) Climax (days) Maximum induration (mm) Duration of maximum response(days)
7 49 to 56 4.0 to 6.5 10 to 14
10 46 3.5 to 5.5 9to 11
10 50 3.5 to 5.0 10 to 12
NR*
8 4.5 to 6.5 9to 15
12 4.0 to 6.0 11 to 15
16 5.0 to 5.5 11 to 13
NR
7 5.0 to 6.5 11 to 16
12 4.0 to 6.0 11 to 18
12 5.0 to 5.5 10 to 14
NR
11 5.5 to 6.5 10 to 17
12 4.0 to 6.0 11 to 17
12 5.0 to 5.5 12 to 15
NR
*NR, nonreactive (controls inoculated with physiological
infection, the fungus was demonstrable for 24 to 27 days, and the lesions healed by the fiftieth day; in the quarternary infection, the fungus was not found after 8 days, but the lesions did not heal for 19 to 21 days. The shortened period of positive skin scrapings is explained by an immune response, and the prolonged period to healing suggests an overlapping of symptoms of infection and hypersensitivity. Histological preparation of the skin lesions verified that the fungi did not penetrate below the comeum stratum, and lay parallel to the epidermal layers. hypersensitivity:
E. floccosum
Controls
saline).
vanbreuseghemii
Delayed
M. vanbreuseghemii
In vivo responses
The results of intradermal challenge of infected animals with test antigens are shown in Table III and Figs. 2 to 4. The onset of a delayed hypersensitivity response coincided with the developmental or early climax stage of the lesions. The maximum response during primary infection was reached at approximately the same time for all three fungi, even though the clinical course varied. These marked differences in infection and reinfection were also absent in the magnitude of the hypersensitivity response to the three fungi. The time to maximal hypersensitivity response in reinfection was considerably shorter than in primary infection, and there was a moderate increase in the size of the indurated area during reinfection.
The persistance of the delayed cutaneous reaction is shown in Figs. 2 to 4. These data show the responses during active infection and after healing of the final experimental infection until the animals became nonreactive. The hypersensitivity diminished over a period of twelve months for T. rubrum and ten months for M. vanbreuseghemii, but it diminished abruptly for E. jloccosum and was absent by six months. The skin test antigen did not interfere with the DHS or with clinical infection. Control uninfected animals inoculated with the three test antigens in parallel with the infected animals did not develop cutaneous DHS responses, and these animals were susceptible to infection when they were inoculated with the fungi after this prolonged exposure to the test antigen. Delayed
hypersensitivity:
In vitro
responses
The results of the migration inhibition assay are shown graphically in Fig. 5. There was a significant difference between the MM1 of controls and the experimental groups (p < 0.05, Student’s t test), and inhibition reached 60% in the case of M. vanbreuseghetnii. The percentage inhibition by T. rubrum antigen was smaller than the response to the other two fungal antigens, but the difference was not significant (p < 0.05, Student’s t test). The results of the LT assays for specific antigenic
VOLUME NUMBER
TABLE
Cell-mediated immune responses to dermatophytes
63 5
IV. Passive
transfer
of cutaneous
sensitivity
in guinea
lntradermal Recipient’ TM Et
ii 2$ 3i: 4$ 5$ 63 I 8 9 10 II c c c c
12 c c
13 14 16 17 c c c c ccc
15 18 c c
19$20$2 I 22$23 241 25 26 21 28 29$30 ccc ccc
31432 334 34335 36; 37 38$39 40 41$42 c c c c c c
Number of ceils transferred
3.0-5.0 x IOR
1.0 x 10”
2.0-2.5 x 108
2.0-2.5 x 108
24 hr TME
46 hr TME
Splenic cells +-+ +-+++ +--
Peripheral +++ +----
9 days
12days
15days
TME
TME
TME
t-+ +++ -+-
f-+ +++ -+-
f-f +++ -+---
+-+++ -+-
-+-
leukocytes +++ +++ +-+-+++ +++ -----
+--
test reaction
6 days TME
Peritoneal exudate c~ells +++ +++ +-++++++-
pigs
72 hr TME
Inguinal lymph node cells +-+-+------+-
++-
367
Positive transfers
T
M
E
2/4 2/4 2/4
---
1/2 l/2 o/2
+++ +--++
-++ +--
3/4 2/4 2/4
+++ ++-
+++ ++-
-++--
++-
-+-
---
+--+-
-+-
-t-t+ +-+++
3/4 3/4 l/4 +--
C, Controls receiving cells from nonsensitized donors. *Animals 1-42 were 2-3 wk old guinea pigs receiving cells from sensitized donors. tT, T. ruhrum; M, M. vanhreuseghemii; E, E. floccosum $The progeny of the donor.
activators are depicted in Fig. 6. In the controls with nonspecific mitogens, the 3H-thymidine uptake of the 1ymphocyl:es of sensitized and nonsensitized animals indicated :significant activation. The response of sensitized animals was not significantly higher than that of the nonsensitized, although it is interesting that lymphocytes from the recently infected animals tended to be more responsive. There was a significant difference in the response of the same lymphocyte populations from the experimental and control groups to the specific antigenic activators. As in the macrophage inhibition assay, the response to T. rubrum antigen was less than the other two antigens, but there was no significant difference between the responses in the three experimental groups (p < 0.05, Student’s t test).
Passive transfer
of sensitivity
The results of the studies on passive transfer of delayed hypersensitivity to the dermatophytes are shown in Table IV. Donor splenic cells and peripheral leukocytes sensitized 50% to 75% of all the recipients. Peritoneal exudate cells sensitized some animals in each group, but only 1 of 4 recipients became responsive to the E. jbxosum antigen. The only donor populations which failed to elicit a response were cells from the inguinal lymph nodes in the case of E. poccosum; however, the lymph node cells sensitized 50% of the animals tested with the other two fungi. The passively transferred cutaneous sensitivity was short-lived and only two animals remained sensitized on the last day of the experiment (fifteenth day).
366
Hunjan and Cronholm
DISCUSSION Infection by dermatophytes undoubtedly may be accompanied by an acquired immune response. This is verified primarily by the extensive documentation that cutaneous sensitivity is a common correlate to infection. The major unresolved issues include the relationship between this sensitization and a predictable alteration in the status of the host to reinfection. This information is essential for an understanding of the basic mechanisms of these diseases, and to any rational program of disease management by immunological methods. These issues are also of theoretical interest because they include the interesting problem of systemic responses to noninvasive pathogens. A single assay for hypersensitivity is inconclusive evidence that the full range of CM1 responses is operative. The direct evidence for CM1 to dermatophytes is found primarily in the reports of local hypersensitivity reactions. This study combined in vitro and in vivo assays for systemic CM1 as correlates to these local reactions. The results of this systematic investigation present compelling evidence that a full array of CM1 is associated with these fungal infections. Clinically, the course of the disease was altered in a pattern suggesting acquired immunity, with a marked condensation of the infectious phases and variations in the lesions, which are reasonably explained as an exacerbation by the inflammatory stages of cutaneous hypersensitivity. This shift in the clinical course upon reinfection of animals was also reported by DeLamater and Benham15 and in humans by Jones, Reinhardt, and Rinaldi .4 The specific course of alteration in reinfection for the three dermatophytes bore an interesting relationship to the primary disease, and verifies the correspondence noted by others between immunity and host range.2 M. vanbreuseghemii, a zoophilic species, elicited a severe inflammatory response in primary disease and reinfection. T. rubrum, which is primarily anthropophilic but is known to infect other species, produced a moderate inflammatory response in the primary infection and a severe inflammation in reinfection. E. poccosum, previously considered obligately anthropophilic, produced only a mild inflammation in all infections. The course of the cutaneous hypersensitivity developing during infection was similar to other reports,’ and indicates that the immune status of our experimental animals was similar to equivalent systems, including humans. There was consistency between the data obtained on persistence of cutaneous hypersensitivity after infection and the clinical pattern in reinfection. The pathogen which elicited the mildest
J. ALLERGY
CLIN. IMMUNOL. MAY 1979
symptoms on primary infection and the least alteration on reinfection also produced a short-lived hypersensitivity. Two major contributions of this study are the MM1 and LT assays, which are important confirmations of the sensitization of systemic immunocompetent cells to the antigens which yield positive skiI! tests. The inhibition of macrophage migration and activation of the lymphocytes by specific antigen should be interpreted as direct evidence for a systemic response to these fungi. The results of the passive tracsfer by cells is less satisfying because syngeneic animals were unavailable, and because the populations of donor cells were mixed. However, these data present valid additive evidence of a systemic CM1 response. The relative magnitude of infection and the LT and MM1 responses were not as consistent as the reinfection and cutaneous hypersensitivity reactions, in that E. jotcosum antigen and E.$occosum-sensitized cells were more reactive than the T. rubrum systems. However, the correspondence with clinical syndrome (and host range) was observed in the relative effectiveness of passive transfer of hypersensitivity by M. vanbreuseghemii- and E. jfoccosum- sensitized cells. This study does not resolve the inconsistencies between the experimental and clinical data on human hosts, which show that the altered immunity found in experimental conditions is not reflected in resistance to natural reinfection. It must be emphasized that such CM1 responses as DHS do not necessarily correlate with resistance. Further, many factors in natural infection, including variations in host immune competence and exposure to cross-reacting antigens, may alter the individual clinical pattern. There might also be differences in the distribution of diffusible antigen as a function of the specific host-parasite relationship. Such variations must be considered within a general model for a CM1 response to dermatophytes that is confirmed by this study. REFERENCES 1. Neisser A: Plato’s versuche uber die Herstellung und Verwendung von “Trichophytin,” Arch Dermatol60:63, 1902. 2. Grappel SF, Bishop CT, Blank F: Immunology of dermatophytes and dermatophytosis, Bacterial Rev 38:222, 1974. 3. Cruickshank CND, Trotter MD, Wood SO: Studies on trichophytin sensitivity, .I Invest Dermatol 35219, 1960. 4. Jones HE, Reinhardt JH, Rinaldi MG: Acquired immunity to dermatophytes, Arch Dermatol 109:840, 1974. 5. Bloch B, Massini R: Studien uber Immunitat und Uberempfindichkeit bei Hyphomycetener Krankungen, Z Hyg Infeksionskr 6368, 1909. 6. Sloper JC: A study of experimental human infections due to Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton j7occosum, with particular reference to the
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self-limitation of the resultant lesions, J Invest Dermatol 25:21, 1955. Jones HE. Reinhardt JH, Rinaldi MG: Model dermatophytosis in naturally infected subjects, Arch Dermatol 110~369, 1974. Kabat EA. Mayer MD: Experimental immunochemistry, Springfield, Ill., 1948, Charles C Thomas, Publishers. David JR, Al-Askari S, Lawrence HS, Thomas L: Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigen, J. lmmunol 93~264, 1964. Jondal M, Holm G, Wigzell H: Surface markers of T and B lymphocytes, J Exp Med 136~207, 1972. Valentine F: Lymphocyte transformation, in Bloom BR, Glade
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PP. editors:/n vim methods in cell mediated immunity, 197 1, Academic Press Inc., New York. Chase M: The cellular transfer of cutaneous hypersensitivity. J Bacterial 51:643, 1946. Haxthausen H: Experiments on passive transfer of eczematous allergy, J Invest Dermatol 19:293, 1952. Howard DH: Fate of Hisroplasma capsulutum in guinea pig polymorphonuclear leucocytes, Infect Immun 8~412, 1973. DeLamater ED, Benham RW: Experimental studies with the dermatophytes. II. Immunity and hypersensitivity produced in laboratory animals, J Invest Dermatol 1:469, 1938.
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