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Human Skin Window
HUMAN SKIN WINDOW A CYTOLOGIC METHOD FOR THE STUDY OF ALLERGIC INFLAMMATION*
FUNAN RU, M.D., ROBERT P. FOSNAUGH, M.D., HENRY G. BRYAN, M.D. AND DIANA JACKS, B.S. The cytology of acute inflammation was originally described by Metchnikoff in experimental animals. Rebuek and Crowley (1) have advanced the technic of human skin windows which confirmed the description of the acute inflammatory cycle. The basic sequences consisted of an early migration of phagocytic granulocytes, reinforcing available tissue macrophages. These phagocytes were supplemented by migrating lymphocytes
The cells of the inflammatory exudate migrated
to the under-surface of the eoverglass flattening out much in the same manner as a blood smear,
thus permitting detailed study of the cellular infiltrate. At regular intervals the eoverglass was removed and rapidly air-dried. At the same time
another sterile eoverglass was placed over the same denuded area and the process was repeated
at designated intervals as often as desired. In
this way, a series of permanent, fixed preparations and blood mononuclears (monocytes) which of in vivo samplings of the inflammatory exudate
hypertrophied to form additional macrophages. was obtained. The eoverslips, after being In the present study we have employed the skin air-dried, were stained like blood smears with the window technic to study the inflammatory re- May-Gruenwald-Giemsa stain. After dehydration and mounting, the specimens were ready for sponse to a contact allergen. cytological examinations. Experiment I. Diphtheria toxoid (Aluminum MATERIALS AND METHODS phosphate adsorbed, Bio. 2110, Parke, Davis and Technic: The skin of the forearm was shaved, Company) was employed as a non-pyogenic cleansed with 70% ethanol and air-dried. By means antigen in patients with various skin diseases, as of a sterile Bard Parker No. 15 scapel blade, the well as in normal volunteers. Simultaneously with epithelium was scraped away from an area of skin the application of the skin window, a Schick or 3 to 4 mm. in diameter until the papillary layer Moloney skin test was performed. Two time of the corium was exposed. Multiple, minute bleed- schedules were chosen for this experiment. ing points were evidence that the corium was (a) The eoverglasses were removed and reached and capillary loops exposed. A drop of the reapplied at 3, 6, 9, 12, 14 and 24 hours. solution or suspension of the desired testing agent (b) The eoverglasses were removed and was applied to the denuded corium. The lesion reapplied at 24, 27, 30, 33, 36, 38, 48, 52, 56, was then immediately covered with a sterile, and 60 hours. chemically clean, 12 mm., No. 2 coverglass, Experiment II. Diluted acetone extract of Rhus which was, in turn, covered by a square of card- oleoresin* (1:200) was used as antigen in patients board of the same size. The eoverglass and card- with atopie dermatitis and in normal volunteers. board were fixed in place by a special surgical tape At the time the window was applied, a patch test (Experimental surgical tape, Scotch Brand, was to Rhus extract 1:1000 was applied to the skin to supplied through the courtesy of Minnesota determine whether the individual was allergic by Mining and Manufacturing Company. This tape contact to Rhus antigen. A peripheral blood smear produced less epithelial damage from repeated was taken at the same time the window test was applications at the same site. It also permitted initiated and a second blood smear was taken at evaporation of skin moisture thus causing less the end of 48 hours. maceration of the skin). The same time schedule was employed in this * From the Department of Dermatology (Clar- experiment as in the experiment 1(a) and 1(b).
ence S. Livingood, M.D., Chairman), Henry
Ford Hospital, Detroit, Michigan. Supported by research grants from the National Institutes of Health, U. S. Public Health Service
RESULTS
Experiment 1(a): Employing diphtheria toxoid (CV 3345) and Medical Department, BristolMyers Products Division, Bristol-Myers Com- as antigen. 3 Hour Stage: About 90% to 95% of the exupany. Presented at the Twenty-second Annual Meet* Rhus oleoresin, courtesy of Allergy Laboraing of The Society for Investigative Dermatology, tories, Hugh Graham, Inc., Dallas, Texas. Inc., New York, N. V., June 27, 1961. 409
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
410
FIGs. 1 TO 5. Inflammatory exudate in windows utilizing Rhus as antigen in Rhus patch test negative individual. FIG. 1. 6 hour stage. Note the predominance of neutrophilic leukocytes. FIG. 2. 9 hour stage. FIG. 3. 12 hour stage. FIG. 4. 24 hour stage. Note the predominance of the mononuclear cells. FIG. 5. One macrophage with ingested nuclear lobes from a neutrophil.
dated cells were ncutrophils. The rest consisted of small lymphocytes, tissue macrophagcs and
still the predominant cell type. The mononuclear
occasional cosinophils.
population. Many of these were small lymphocytes (Fig. 1).
6 Hour Stage: Ncutrophilic lcukocytes were
cells made up about j4 to
of the total cell
411
HUMAN SKIN WINDOW
too 90 -
9 Hour Stage: The neutrophils at this time were
reduced to constitute oniy one-half of the total
cell population. In addition to decreasing in numbers, the neutrophils had become shrunken and their nuclei darkly stained. This was in direct contrast to their large and edematous appearance at the 3 hour samples (Fig. 2). The lymphocytes increased greatly in number, now constituting approximately 50% of the total cell population of the inflammatory exudate. Most were relatively small lymphocytes, similar to those seen in the peripheral blood smear, but some had begun to show hypertrophie changes, taking on the appearance of maerophage-like cells. These cells also showed some evidence of phagoeytic activity. As time went on, the percentage of the hyper-
80 70
R 60 C
so
E N
I 30 20 I0 I
trophied lymphocytes and macrophages increased, while that of the small lymphocytes
I
I
I
I
I
I
I
3 6 9 1215 1821 24 RHUS POSITIVE 3-24 HOURS
decreased (Fig. 3). The neutrophils steadily decreased in number,
00
constituting approximately 3/ to }4 of the total
cell population, and showed evidence of de-
90
generative changes.
24 Hour Stage: Almost all cells were of the mononuclear type, consisting essentially of macrophages and hypertrophied lymphocytes (Fig. 4). Only occasional neutrophils were present at this stage, and most of these showed evidence of degenerative changes in the form of pyknotie and disintegrating nuclei. Some nuclear remnants were seen inside of macrophages (Fig. 5). Our results in this experiment correspond well to the observations originally reported by Rebuck
and Crowley (1). There was no apparent difference in the cellular exudation in the inflamma-
tory cycles in the Schick positive and Schick
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40 T 30
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20-
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negative individuals included in this study. n. 0 I I I Experiment 1(b): Employing diphtheria toxoid 3 6 9 12 15 18 21 24 as antigen. RHUS NEGATIVE 3-24 HRS. Three normal volunteers were tested in this 6 Fins. AND 7. Curves showing the distribution experiment. One of the individuals had a nega- and percentage of inflammatory cells in windows tive Moloney test; one had a markedly positive taken at 3 to 24 hour periods. Fin. 6. From a Rhus-positive individual. Moloney and one had a markedly positive Schick Fin. 7. From a Rhus-negative individual. test. —— Neutrophils, In the Moloney-negative subject there was a Lymphocytes, Macrophages. wave of neutrophilic migration to the inflammatory site at the 27 to 33 hour stages (a similar wave of neutrophilic migration at 3—6 hours oc- neutrophils while the number of lymphocytes curred in Experiment 1(a)). There was still remained relatively constant, varying from 5% another wave of neutrophilic migration at 50 to to 10%. In the Schick-positive and Moloney-positive 56 hours. In general the percentage of macrophages fluctuated inversely with that of the individuals the neutrophil response at the 3-hour I
412
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE I Patch test and skin window results in non-atopic individuals Subject
Diagnosis
Patch Test to Rhus
1
Normal Normal Normal Normal Chronic dermatitis Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Stasis dermatitis Chronic dermatitis Normal Normal
Positive Positive Positive Positive Positive Positive Weakly positive Weakly positive Weakly positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
2 3 4 5 6 7 S
9 10 11 12 13 14 15 16 17 18 19 20 21
Stasis ulcer
''e
Bacteria
X X )<
X X X X
X
X
X
X X X
X X X
x
X
x
X X X X X X X X X
stage was 45% to 55%, while that of the patch test and nine showed a positive patch test to Rhus at 1:1000 dilution. The findings are positive individuals had a slight increase in summarized in Table I. A cell distribution curve Moloney-negative individual was at 85%. Both
from a highly Rhus-sensitive, normal volunteer is illustrated in Figure 8. Another from a Rhusnegative individual is illustrated in Figure 9. 24—33 Hour Stage: There was no significant age of lymphocytes in the positive than the differences between the windows of the patch negative individuals. The significance of the test positive and negative individuals at this eosinophils in the Schick and Moloney positive stage. An influx of neutrophils to the inflammaindividuals is not known at the present time. tory field usually occurred at the 27—30 hour stage. While the percentage of the hypertrophied Experiment II: Utilizing Rhus antigen. The subjects in this experiment were divided lymphocytes and macrophages fluctuated ininto patch test-positive and patch test-negative versely with that of the neutrophils, the lymphocytes appeared to remain in a more or less congroups. (a) During the first 24 hours there was essen- stant low level, i.e., between 5% and 10%. tially no difference in the skin windows between (Fig. 10, 14) the positive and negative individuals. The per33—48 Hour Stage: Differences between Rhuscentages of the different cell types seen in the positive and Rhus-negative began to appear windows corresponded closely to those observed after the 33 hour stage. (Fig. 11, 12, 13, 15, 16, eosinophils at this stage, varying from 4% to 8%. The eosinophils were not found in the Moloneynegative individual. There seemed to be a slightly greater percent-
during the first 24 hours utilizing diphtheria
toxoid (Fig. 1, 2, 3, 4). A typical Rhus-positivc
17, 18).
(1) Eosinophils appeared in increasing numbers in the Rhus-positive subjects, varying from curve arc illustrated in Figures 6 and 7. 10% to 40%; eosinophils were either absent or (b) Twenty-one persons were included in this present in insignificant numbers in Rhus-negaexperiment, among them 12 showed a negative tive individuals.
and a typical Rhus-negative cell distribution
HUMAN SKIN WINDOW
Fl
100
cells were found in one normal volunteer who had
90
a severe reaction to Rhus extract at the patch test and window sites (Fig. 17). This individual developed an urticarial reaction 72 to 84 hours after exposures to Rhus. nbc reaction lasted for about three days. (3) The neutrophils in the positive windows tended to remain relatively high at the beginning of this stage, gradually declining to a low level
60 50
N 40
as time passed. In the negative windows the
T 30
neutrophils exhibited more frequent fluctuation
in numbers as shown in the cell distribution
20
curve in Figure 9.
(4) The lymphocytes in the negative indi-
10
0
viduals remained at the same low level as semi 24 27 30333639 RHUS POSITIVE
24-60 HOURS
tOO-
90-
R
E
413
60 50
N 40
T 30 20
in the 24—33 hour stage. In the positive windows there was an increase in the number of lymphocytes at approximately the same time the eosinophils began to increase.
(5) In negative windows the percentage of macrophages appeared to fluctuate in the opposite direction from the neutrophils, the lympho-
cytes remaining more or less constant. In the windows of the Rhus-negative individuals the number of the hypertrophied lymphocytes and macrophages appeared to be more numerous. Also, these cells appeared to be better preserved in the later hours (Fig. 13). This was in contrast to the inflammatory exudate in the positive sub-
jects, which was cluttered with cellular debris (Fig. 17). 48—60 Hour Stage: The picture in these stages
IC.
showed essentially an extension of the processes observed in the 48 hour stage. There were more 24273033363942454851 545760 degenerative changes in both Rhus-positive and Rhus-negative window. A new wave of neutroRHUS NEGATIVE 24-60 HRS. phils was often seen at 52 and 56 hour stage in Fios. 8 AND 9. Curves in windows taken at 24 the Rhus-negative windows. to 60 hour period. (c) The subjects in this group were either paFIG. 8. From a Rhus-positive individual. Fm. 9. From a Ithus-negative individual. tients with classical atopic dermatitis or paNeutrophils. tients exhibiting other evidence of atopy. There Lymphocytes were eight patients in this group. Four reacted Macrophages. —. . . — Eosinophils. positively and two reacted negatively by patch Vertical Lines. — — — — Appearance of vesi- test to 1:1000 Rhus antigen; two reacted posides on intact skin surrounding the deuuded Appearance of multinu- tively only to 1:200 Rhus antigen. The inflamwindow site. cleated giant cells. matory patterns in this group resembled someIn.
what those of Rhus-negative, non-atopic indi(2) The appearance of bi- and tn-nucleated viduals. The results are summarized in Table II. cells occurred in the later hours of these stages. In all of our subjects there was no change in These cells were present in both the negative the peripheral differential counts in the blood and positive windows. Large multi-nucleated, smears taken at the beginning of the experiment giant cells with five to thirteen nuclei were seen and at 48 hours. None of the subjects showed only in Rhus-positive windows. The largest giant
evidence of blood eosinophilia.
414
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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FIGs. 10 TO 15. Inflammatory exudate in Rhus-negative windows during the 24 to 60 hour period. FIG. 10. 24 hour stage. Note the predominance of the mononuclear cells. FIG. 11. 36 hour stage. FIG. 12. 38 hour stage. FIG. 13. 48 hour stage. Note the macrophages with engulfed nuclear remnants of degenerated neutrophils. FIG. 14. 24 hour stage. FIG. 15. 36 hour stage.
HUMAN SKIN WINDOW
415
FIGs. 16 TO 19. InflammatGry exudatG in Rhus-positive windows during the 38 to 48 hour period.
FIG. 16. 38 hour stage. Note the presence of eosinophils (Arrows). FIG. 17. 48 hour stage. Note the large multinucleated giant cell. Only small number of macrophages were present. The neutrophils appeared shrunken with pyknotic nuclei. FIG. 18. Eosinophils at 48 hour stage in Rhus-positive window. FIG. 19. Leukocytes with ingested bacteria. DISCUSSION
tive subject. It is our intention to do further
Our results, utilizing diphtheria toxoid in the studies along these lines. During the first 24 hours, utilizing Rhus as skin windows (Experiment 1(a)), correspond well to the original observations of Rebuck and antigen in the skin windows (Experiment 11(a)), Crowley (1). There was no apparent difference in there was essentially no difference between the inflammatory cellular exudates between Rhus-positive and Rhus-negative subjects. The Schick-positive and Schick-negative individuals differential counts of the inflammatory exudates in windows taken during the first 24 hours. In in the preparations taken during this period the 24—60 hour windows, utilizing diphtheria correspond closely to those observed during the toxoid as antigen (Experiment 1(b)), there ap- first 24 hours using diphtheria toxoid as antigen. The most striking feature in the Rhus-positive peared to be some difference in the inflammatory response between Schick or Moloney-positive windows was the presence of eosinophils at 33—48 and Moloney-negative indivuals. In the prepara- hour stage. These cells were never found in such tion taken from Moloney or Schick-positive sub- enormous numbers in any of the individuals who jects, there were consistently a few eosinophils had negative patch test to Rhus antigen. The eosinophils appeared in the inflammatory present; this was not true in the Moloney-nega-
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
exudate about the same time that vesiculation
nation (Fig. 19, 20). None of the windows taken
was evident clinically in the intact skin surrounding the denuded area. The presence of eosinophils
from Rhus-positive individuals showing the
sensitization to Rhus oleoresin and their absence in windows from subjects who are non-reactive to Rhus suggest some basic difference in reaction
The inflammatory responses seen in windows of Rhus-negative subjects from 24 to 60 hours are
eosinophilie response showed evidence of bacin the windows from subjects having contact terial contamination.
pattern between allergic and non-allergic individuals. However, it should be noted that not all of our Rhus-positive subjects exhibited this eosinophilic response. The majority of these Rhus-positive subjects exhibiting atypical response had only weakly positive patch test to Rhus antigen (Table I). It appears reasonable to assume that a weak patch test reaction represents a lesser degree of sensitization.
An additional finding in the windows taken from Rhus-positive individuals exhibiting atypical inflammatory reaction (lack of eosinophils) was the invariable presence of bacterial contami-
represented in Figure 9. These inflammatory curves are characterized by repeated waves of large numbers of neutrophils. Between the waves
of neutrophils there appears to be an influx of maerophages. The percentage of the lymphocytes remains more or less constant. It should be noted that this response differs in some cases from the
atypical response seen in Rhus-positive individuals. Our findings in Rhus-positive, atopie individuals were indeed interesting. Preparations taken
from these patients did not demonstrate the eosinophilia present in our strongly Rhus-posi-
tive, non-atopie subjects and therefore the inflammatory curves are somewhat similar to our Rhus-negative, non-atopie individuals. Evidence of bacterial contamination was present in all but
two of our atopie subjects. The large multinucleated "epithelioid" giant cells were also absent.
It is not known whether the absence of the eosinophilic response in our atopic subjects is another example of the atopic "paradox" or due to bacterial contamination. The significance of the large multinucleated "epithelioid" giant cells which appeared during the later hours (from 48 to 60 hours) only in the Rhus-positive subjects is not known. BraunFia. 20. A mononuclear cell with ingested bac- steiner et al (2) who performed window experiteria. This probably represents a lymphocyte ments on patients utilizing tularin or tuberculin which exhibits phagoeytic activity. TABLE II Patch test and skin window results in atopic individuals Subject
Diagnosis
Patcb Test to Rbus
1
Atopie dermatitis* Atopic dermatitis Atopie dermatitis
Weakly positivet Weakly positive Positive Positive Positive Positive Negative Negative
2 3
4 5 6
Atopie
7
Atopie Atopie
8
Atopie
Atopie
Typical
Response
* Classical atopic dermatitis along with other evidence of atopy.
t Positive only at llhus extract 1:200 but negative to 1:1000. t Atopie stigmata but without classical atopie dermatitis.
Atypical Response
nactecia
x
x
X X X X X X
X X X
X
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HUMAN SKIN WINDOW
417
as antigen found massive infiltration of lympho- tionship between the presence of eosinophils and cytes which rapidly hypertrophied and became immune responses. In further studies be (8) con"mononuclear", "epithelioid", and even giant cluded that the eosinophilic response to injections cells. These derivatives of lymphocytes were of antigens paralleled antibody production. found to be capable of transferring delayed skin Torre and Leikin (9) performed window tests allergy in a highly specific manner. In this study, on patients with eosinopbilia. In their experithe authors found that the eosinophils were ments there were large numbers of eosinophils rather scarce in the exudate even in the presence during the early hours of the experiment. The of blood eosinophilia. Rebuck and Yates (3) also eosinopbils gradually decreased in number in noted the presence of large macrophages and subsequent windows but never completely disgiant cells in the tuberculin-positive windows at appeared. 63 hours. Riis (10) found no regular correlation between Rebuck and Yates (3) studied the cellular re- tbe number of eosinophilic granuloeytes in the sponses in windows in tuberculin-negative and peripheral blood and in the inflammatory exupositive subjects, employing Old Tuberculin as date. antigen in these studies. The inflammatory reCONCLU5ION5 sponses in subjects with negative tuberculin tests were similar to the responses to other nonpyo1. The skin window offers a dynamic approach genie antigens, i.e., diphtheria toxoid. Marked for studying the inflammatory exudate produced alterations in the neutrophilie sequences occurred by various types of antigens, irritants and in the tuberculin-positive subjects. These altera- trauma. The application of diphtheria toxoid tions resulted in defective and retarded mono- under the skin window produces inflammatory nuclear responses which were primarily a reflec- reaction of a nonpyogenic nature and serves as a tion of lymphocytic depression in the inflamma- proteotype or baseline for comparing other types tory cycle. The importance of a normal neutro- of stimulation. 2. Within the first 24 hours a skin window, philic response was further confirmed by Page and Good (4, 5) in an extensive study of inflam- utilizing diphtheria toxoid as the test substance, mation in a patient with cyclic neutropenia and will show early migration of pbagoeytic granuloin rabbits with experimentally induced neutro- cytes which are later supplemented by migrating
penia. They demonstrated that the absence of lymphocytes and blood monunuelears. In still neutrophils from the circulation profoundly later stages these mononuclear cells have been alters the early events of the inflammatiory cycle. Our experiment appears to illustrate the basic difference between tuberculin and contact ecze-
matous sensitivity. The lack of significant difference in the first 24-hour windows of Rhuspositive and Rhus-negative subjects is in direct contrast with what occurs in tuberculin allergy. The characteristic reaction patterns in the Rhuspositive windows did not become evident until
shown to hypertrophy to form additional macrophages to supplement tissue maerophages. 3. When Rhus oleoresin was applied under a skin window no difference in inflammatory pat-
tern between individuals positive by patch test to Rhus and individuals negative by patch test to Rhus was evident until the 33 hour stage. The Rhus-positive, aon-atopie individuals differed from the negative reactors as follows: (a) Eosino-
33 hours.
philia, (b) larger number of lymphocytes, and Eitzman and Smith (6), employing the skin (c) the presence of large multinueleated giant
window technic, described eosinophilic response cells. in newborn infants from two to twenty-one days 4. Bacterial contamination appeared to modify after delivery. The eosinophils appeared in the inflammatory pattern in the skin windows. windows from two hours to twelve hours. There 5. Atopic subjects appeared to react somewhat was no consistent high blood eosinophilia in these subjects.
differently from non-atopic, "normal" individuals.
Speirs (7) noted the accumulation of eosinoACKNOWLEDGMENTS phils between the second and the tenth day after The authors gratefully acknowledge the advice repeated injections of antigenie materials. His experiments suggested the existence of the rela- and assistance from Dr. J. W. Rebuck of the
418
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Department of Laboratories in the early phase of this study. REFERENCES 1. REBUCK, J. W. AND CROWLEY, J. H.: A method
experimental study of the function of neu-
trophils in the inflammatory response.
Amer. J. Path., 34: 645—669, 1958. 6. EITZMAN, D. V. AND SMITH, R. T.: The non-
specific inflammatory cycle in the neonated infant. A.M.A. J. Dis. Child., 97: 326—334, 1959.
of studying leukocytic functions in vivo.
7. SI'EIRS, R. S.: Physiological approaches to an
2. BRAUN5TEINER, H., PAERTAN, J. AND THUMB,
phils and basophils. Ann. N. Y. Acad. Sci.,
Blood, 13: 417—426, 1958. 3. REBUCIC, J. W. AND YATES, J. L.: The cytology
of the tuberculin reaction in skin windows
8. SPEIE5, R. S.: Advances in the knowledge of the eosinophil in relation to antibody formation. Ann. N. Y. Acad. Sci., 73: 283—306,
226, 1954.
9. TOREE, C. M. AND LEIKIN, S. L.: Tissue in-
Aon. N. Y. Acad. Sci., 59: 757—805, 1955.
N.: Studies on lymphocytic functions. in man. Amer. Rev. Tuberculosis, 69: 216—
4.
PAGE, A. R. AND GooD, R. A.: Studies on cyclic neutropenia. A.M.A. J. Dis. Child., 94: 623—661, 1957.
5. PAGE, A. R. AND Gooo, R. A.: A clinical and
understanding of the function of eosino59: 705—731, 1955.
1958.
flammatory cytology in clinical states. Amer. J. Chn. Path., 32: 335—345, 1959. 10. Rt's, P.: The Cytology of Inflammatory Exudate. Copenhagen. Munksgaard, 1959.
DISCUSSION Dn. ARTHUR W. GRACE (New York, New York): I was rather uncertain about the column headed "Bacteria" in Tables No. 1 and No. 2. It wasn't clear to me whether we are dealing with blister fluids in there or not. It has been my experience with spontaneously appearing blister fluids or those that were induced by the use of
to identify the bacteria in the windows. We
blister fluids remain bacteriologically sterile for about twenty-four to thirty-six hours after their formation. At what time after the appearance of such blisters as there were in these two tables, was the study for bacteria made known? Were the bacteria identified only by direct observation through a window or by direct observation and by staining?
as 12 hours after the initiation of the window. Employing the May-Gruenwald-Giemsa stain, the bacteria can be seen to increase in number from hour to hour in the phagocytic cells. There is no evidence of clinical infection even though the windows show bacterial contamination microscopically. In the Rhus-sensitive individuals, the denuded sites actually heal faster than the
DR. ROBERT W. GOLTI (Minneapolis, Minne-
intact skin around it. The unabraded skin around
believe that these organisms are normal resident as well as transient skin flora. Since the window sites arc sterilized only by superficial cleansing with alcohol before scraping, it would be expected
that some of the bacteria of the skin left behind
in the follicles eventually may move into the cantharides paste on normal skin, that such inflammatory site. We see these bacteria as early
sota): Dr. Fa,saro and Dr. Good of the Uni- the window eventually shows symptoms and versity of Mii;aesota have carried out this technic
over the past year or two. Their particular interest centered around dermatitis herpetiformis; and perhaps I shouldn't quote their results without consulting with them but, as I remember it, they showed an increase in cosinophil infiltration in the very early stages within the first twentyfour hours, in contrast to Dr. Punan Ru's findings.
signs of a classical contact dermatitis to Rhus. In response to Dr. Goltz's question and comments on dermatitis herpetiformis, we have done
skin windows in this condition, but we are not as yet ready to report our findings. We do see eosinophils appearing in the windows in the early
stages when the patients have a high blood eosinophil count. We consider this a nonspecific response. There are several reports of the cosino-
four hours, however, feeling that by that time
philic response in windows. The correlation between blood eosinophilia and tissue eosino-
bacterial contamination had become such a factor
philia vary so much that most observers consider
They gave up their observations after twenty-
that the observations were no longer reliable it non-significant. A high blood cosinophilia after that time. Perhaps they should have gone apparently may or may not be accompanied by on further. a high cosinophilic response in the windows. DR. FUNAN Ru (in closing): With regard to In all our experimental subjects, we do a difDr. Grace's question, we have made no attempt ferential white blood cell count at the beginning
HUMAN SKIN WINDOW
419
and at the end of 48 hours of the window experi- hers, and reach a peak from 48 to 55 hours. In ment. None of them shows any significant change contrast to the appearance of cosinophils in the of blood picture in the two specimens. first 24 hours which we consider as a non-specific In the windows of the Rhus-positive indi- response, the eosinophilic response in the Rhusviduals, the eosinophils can go up to as high as positive windows at the late hours, probably is 40 per cent. The eosinophils begin to appear at significant. This response is not found in persons 30 to 33 hour stage, gradually increase in num- with negative patch test to Rhus.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
FUNAN RU, M.D., ROBERT P. FOSNAUGH, M.D., HENRY G. BRYAN, M.D. AND DIANA JACKS, B.S. The cytology of acute inflammation was originally described by Metchnikoff in experimental animals. Rebuek and Crowley (1) have advanced the technic of human skin windows which confirmed the description of the acute inflammatory cycle. The basic sequences consisted of an early migration of phagocytic granulocytes, reinforcing available tissue macrophages. These phagocytes were supplemented by migrating lymphocytes
The cells of the inflammatory exudate migrated
to the under-surface of the eoverglass flattening out much in the same manner as a blood smear,
thus permitting detailed study of the cellular infiltrate. At regular intervals the eoverglass was removed and rapidly air-dried. At the same time
another sterile eoverglass was placed over the same denuded area and the process was repeated
at designated intervals as often as desired. In
this way, a series of permanent, fixed preparations and blood mononuclears (monocytes) which of in vivo samplings of the inflammatory exudate
hypertrophied to form additional macrophages. was obtained. The eoverslips, after being In the present study we have employed the skin air-dried, were stained like blood smears with the window technic to study the inflammatory re- May-Gruenwald-Giemsa stain. After dehydration and mounting, the specimens were ready for sponse to a contact allergen. cytological examinations. Experiment I. Diphtheria toxoid (Aluminum MATERIALS AND METHODS phosphate adsorbed, Bio. 2110, Parke, Davis and Technic: The skin of the forearm was shaved, Company) was employed as a non-pyogenic cleansed with 70% ethanol and air-dried. By means antigen in patients with various skin diseases, as of a sterile Bard Parker No. 15 scapel blade, the well as in normal volunteers. Simultaneously with epithelium was scraped away from an area of skin the application of the skin window, a Schick or 3 to 4 mm. in diameter until the papillary layer Moloney skin test was performed. Two time of the corium was exposed. Multiple, minute bleed- schedules were chosen for this experiment. ing points were evidence that the corium was (a) The eoverglasses were removed and reached and capillary loops exposed. A drop of the reapplied at 3, 6, 9, 12, 14 and 24 hours. solution or suspension of the desired testing agent (b) The eoverglasses were removed and was applied to the denuded corium. The lesion reapplied at 24, 27, 30, 33, 36, 38, 48, 52, 56, was then immediately covered with a sterile, and 60 hours. chemically clean, 12 mm., No. 2 coverglass, Experiment II. Diluted acetone extract of Rhus which was, in turn, covered by a square of card- oleoresin* (1:200) was used as antigen in patients board of the same size. The eoverglass and card- with atopie dermatitis and in normal volunteers. board were fixed in place by a special surgical tape At the time the window was applied, a patch test (Experimental surgical tape, Scotch Brand, was to Rhus extract 1:1000 was applied to the skin to supplied through the courtesy of Minnesota determine whether the individual was allergic by Mining and Manufacturing Company. This tape contact to Rhus antigen. A peripheral blood smear produced less epithelial damage from repeated was taken at the same time the window test was applications at the same site. It also permitted initiated and a second blood smear was taken at evaporation of skin moisture thus causing less the end of 48 hours. maceration of the skin). The same time schedule was employed in this * From the Department of Dermatology (Clar- experiment as in the experiment 1(a) and 1(b).
ence S. Livingood, M.D., Chairman), Henry
Ford Hospital, Detroit, Michigan. Supported by research grants from the National Institutes of Health, U. S. Public Health Service
RESULTS
Experiment 1(a): Employing diphtheria toxoid (CV 3345) and Medical Department, BristolMyers Products Division, Bristol-Myers Com- as antigen. 3 Hour Stage: About 90% to 95% of the exupany. Presented at the Twenty-second Annual Meet* Rhus oleoresin, courtesy of Allergy Laboraing of The Society for Investigative Dermatology, tories, Hugh Graham, Inc., Dallas, Texas. Inc., New York, N. V., June 27, 1961. 409
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
410
FIGs. 1 TO 5. Inflammatory exudate in windows utilizing Rhus as antigen in Rhus patch test negative individual. FIG. 1. 6 hour stage. Note the predominance of neutrophilic leukocytes. FIG. 2. 9 hour stage. FIG. 3. 12 hour stage. FIG. 4. 24 hour stage. Note the predominance of the mononuclear cells. FIG. 5. One macrophage with ingested nuclear lobes from a neutrophil.
dated cells were ncutrophils. The rest consisted of small lymphocytes, tissue macrophagcs and
still the predominant cell type. The mononuclear
occasional cosinophils.
population. Many of these were small lymphocytes (Fig. 1).
6 Hour Stage: Ncutrophilic lcukocytes were
cells made up about j4 to
of the total cell
411
HUMAN SKIN WINDOW
too 90 -
9 Hour Stage: The neutrophils at this time were
reduced to constitute oniy one-half of the total
cell population. In addition to decreasing in numbers, the neutrophils had become shrunken and their nuclei darkly stained. This was in direct contrast to their large and edematous appearance at the 3 hour samples (Fig. 2). The lymphocytes increased greatly in number, now constituting approximately 50% of the total cell population of the inflammatory exudate. Most were relatively small lymphocytes, similar to those seen in the peripheral blood smear, but some had begun to show hypertrophie changes, taking on the appearance of maerophage-like cells. These cells also showed some evidence of phagoeytic activity. As time went on, the percentage of the hyper-
80 70
R 60 C
so
E N
I 30 20 I0 I
trophied lymphocytes and macrophages increased, while that of the small lymphocytes
I
I
I
I
I
I
I
3 6 9 1215 1821 24 RHUS POSITIVE 3-24 HOURS
decreased (Fig. 3). The neutrophils steadily decreased in number,
00
constituting approximately 3/ to }4 of the total
cell population, and showed evidence of de-
90
generative changes.
24 Hour Stage: Almost all cells were of the mononuclear type, consisting essentially of macrophages and hypertrophied lymphocytes (Fig. 4). Only occasional neutrophils were present at this stage, and most of these showed evidence of degenerative changes in the form of pyknotie and disintegrating nuclei. Some nuclear remnants were seen inside of macrophages (Fig. 5). Our results in this experiment correspond well to the observations originally reported by Rebuck
and Crowley (1). There was no apparent difference in the cellular exudation in the inflamma-
tory cycles in the Schick positive and Schick
R
60
/
/ ___ I /
50
40 T 30
//
N
I
,
II I
20-
/I
10-
t
e
negative individuals included in this study. n. 0 I I I Experiment 1(b): Employing diphtheria toxoid 3 6 9 12 15 18 21 24 as antigen. RHUS NEGATIVE 3-24 HRS. Three normal volunteers were tested in this 6 Fins. AND 7. Curves showing the distribution experiment. One of the individuals had a nega- and percentage of inflammatory cells in windows tive Moloney test; one had a markedly positive taken at 3 to 24 hour periods. Fin. 6. From a Rhus-positive individual. Moloney and one had a markedly positive Schick Fin. 7. From a Rhus-negative individual. test. —— Neutrophils, In the Moloney-negative subject there was a Lymphocytes, Macrophages. wave of neutrophilic migration to the inflammatory site at the 27 to 33 hour stages (a similar wave of neutrophilic migration at 3—6 hours oc- neutrophils while the number of lymphocytes curred in Experiment 1(a)). There was still remained relatively constant, varying from 5% another wave of neutrophilic migration at 50 to to 10%. In the Schick-positive and Moloney-positive 56 hours. In general the percentage of macrophages fluctuated inversely with that of the individuals the neutrophil response at the 3-hour I
412
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE I Patch test and skin window results in non-atopic individuals Subject
Diagnosis
Patch Test to Rhus
1
Normal Normal Normal Normal Chronic dermatitis Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Stasis dermatitis Chronic dermatitis Normal Normal
Positive Positive Positive Positive Positive Positive Weakly positive Weakly positive Weakly positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
2 3 4 5 6 7 S
9 10 11 12 13 14 15 16 17 18 19 20 21
Stasis ulcer
''e
Bacteria
X X )<
X X X X
X
X
X
X X X
X X X
x
X
x
X X X X X X X X X
stage was 45% to 55%, while that of the patch test and nine showed a positive patch test to Rhus at 1:1000 dilution. The findings are positive individuals had a slight increase in summarized in Table I. A cell distribution curve Moloney-negative individual was at 85%. Both
from a highly Rhus-sensitive, normal volunteer is illustrated in Figure 8. Another from a Rhusnegative individual is illustrated in Figure 9. 24—33 Hour Stage: There was no significant age of lymphocytes in the positive than the differences between the windows of the patch negative individuals. The significance of the test positive and negative individuals at this eosinophils in the Schick and Moloney positive stage. An influx of neutrophils to the inflammaindividuals is not known at the present time. tory field usually occurred at the 27—30 hour stage. While the percentage of the hypertrophied Experiment II: Utilizing Rhus antigen. The subjects in this experiment were divided lymphocytes and macrophages fluctuated ininto patch test-positive and patch test-negative versely with that of the neutrophils, the lymphocytes appeared to remain in a more or less congroups. (a) During the first 24 hours there was essen- stant low level, i.e., between 5% and 10%. tially no difference in the skin windows between (Fig. 10, 14) the positive and negative individuals. The per33—48 Hour Stage: Differences between Rhuscentages of the different cell types seen in the positive and Rhus-negative began to appear windows corresponded closely to those observed after the 33 hour stage. (Fig. 11, 12, 13, 15, 16, eosinophils at this stage, varying from 4% to 8%. The eosinophils were not found in the Moloneynegative individual. There seemed to be a slightly greater percent-
during the first 24 hours utilizing diphtheria
toxoid (Fig. 1, 2, 3, 4). A typical Rhus-positivc
17, 18).
(1) Eosinophils appeared in increasing numbers in the Rhus-positive subjects, varying from curve arc illustrated in Figures 6 and 7. 10% to 40%; eosinophils were either absent or (b) Twenty-one persons were included in this present in insignificant numbers in Rhus-negaexperiment, among them 12 showed a negative tive individuals.
and a typical Rhus-negative cell distribution
HUMAN SKIN WINDOW
Fl
100
cells were found in one normal volunteer who had
90
a severe reaction to Rhus extract at the patch test and window sites (Fig. 17). This individual developed an urticarial reaction 72 to 84 hours after exposures to Rhus. nbc reaction lasted for about three days. (3) The neutrophils in the positive windows tended to remain relatively high at the beginning of this stage, gradually declining to a low level
60 50
N 40
as time passed. In the negative windows the
T 30
neutrophils exhibited more frequent fluctuation
in numbers as shown in the cell distribution
20
curve in Figure 9.
(4) The lymphocytes in the negative indi-
10
0
viduals remained at the same low level as semi 24 27 30333639 RHUS POSITIVE
24-60 HOURS
tOO-
90-
R
E
413
60 50
N 40
T 30 20
in the 24—33 hour stage. In the positive windows there was an increase in the number of lymphocytes at approximately the same time the eosinophils began to increase.
(5) In negative windows the percentage of macrophages appeared to fluctuate in the opposite direction from the neutrophils, the lympho-
cytes remaining more or less constant. In the windows of the Rhus-negative individuals the number of the hypertrophied lymphocytes and macrophages appeared to be more numerous. Also, these cells appeared to be better preserved in the later hours (Fig. 13). This was in contrast to the inflammatory exudate in the positive sub-
jects, which was cluttered with cellular debris (Fig. 17). 48—60 Hour Stage: The picture in these stages
IC.
showed essentially an extension of the processes observed in the 48 hour stage. There were more 24273033363942454851 545760 degenerative changes in both Rhus-positive and Rhus-negative window. A new wave of neutroRHUS NEGATIVE 24-60 HRS. phils was often seen at 52 and 56 hour stage in Fios. 8 AND 9. Curves in windows taken at 24 the Rhus-negative windows. to 60 hour period. (c) The subjects in this group were either paFIG. 8. From a Rhus-positive individual. Fm. 9. From a Ithus-negative individual. tients with classical atopic dermatitis or paNeutrophils. tients exhibiting other evidence of atopy. There Lymphocytes were eight patients in this group. Four reacted Macrophages. —. . . — Eosinophils. positively and two reacted negatively by patch Vertical Lines. — — — — Appearance of vesi- test to 1:1000 Rhus antigen; two reacted posides on intact skin surrounding the deuuded Appearance of multinu- tively only to 1:200 Rhus antigen. The inflamwindow site. cleated giant cells. matory patterns in this group resembled someIn.
what those of Rhus-negative, non-atopic indi(2) The appearance of bi- and tn-nucleated viduals. The results are summarized in Table II. cells occurred in the later hours of these stages. In all of our subjects there was no change in These cells were present in both the negative the peripheral differential counts in the blood and positive windows. Large multi-nucleated, smears taken at the beginning of the experiment giant cells with five to thirteen nuclei were seen and at 48 hours. None of the subjects showed only in Rhus-positive windows. The largest giant
evidence of blood eosinophilia.
414
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
•S. flatrçw PS -I
&
a;,
FIGs. 10 TO 15. Inflammatory exudate in Rhus-negative windows during the 24 to 60 hour period. FIG. 10. 24 hour stage. Note the predominance of the mononuclear cells. FIG. 11. 36 hour stage. FIG. 12. 38 hour stage. FIG. 13. 48 hour stage. Note the macrophages with engulfed nuclear remnants of degenerated neutrophils. FIG. 14. 24 hour stage. FIG. 15. 36 hour stage.
HUMAN SKIN WINDOW
415
FIGs. 16 TO 19. InflammatGry exudatG in Rhus-positive windows during the 38 to 48 hour period.
FIG. 16. 38 hour stage. Note the presence of eosinophils (Arrows). FIG. 17. 48 hour stage. Note the large multinucleated giant cell. Only small number of macrophages were present. The neutrophils appeared shrunken with pyknotic nuclei. FIG. 18. Eosinophils at 48 hour stage in Rhus-positive window. FIG. 19. Leukocytes with ingested bacteria. DISCUSSION
tive subject. It is our intention to do further
Our results, utilizing diphtheria toxoid in the studies along these lines. During the first 24 hours, utilizing Rhus as skin windows (Experiment 1(a)), correspond well to the original observations of Rebuck and antigen in the skin windows (Experiment 11(a)), Crowley (1). There was no apparent difference in there was essentially no difference between the inflammatory cellular exudates between Rhus-positive and Rhus-negative subjects. The Schick-positive and Schick-negative individuals differential counts of the inflammatory exudates in windows taken during the first 24 hours. In in the preparations taken during this period the 24—60 hour windows, utilizing diphtheria correspond closely to those observed during the toxoid as antigen (Experiment 1(b)), there ap- first 24 hours using diphtheria toxoid as antigen. The most striking feature in the Rhus-positive peared to be some difference in the inflammatory response between Schick or Moloney-positive windows was the presence of eosinophils at 33—48 and Moloney-negative indivuals. In the prepara- hour stage. These cells were never found in such tion taken from Moloney or Schick-positive sub- enormous numbers in any of the individuals who jects, there were consistently a few eosinophils had negative patch test to Rhus antigen. The eosinophils appeared in the inflammatory present; this was not true in the Moloney-nega-
416
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
exudate about the same time that vesiculation
nation (Fig. 19, 20). None of the windows taken
was evident clinically in the intact skin surrounding the denuded area. The presence of eosinophils
from Rhus-positive individuals showing the
sensitization to Rhus oleoresin and their absence in windows from subjects who are non-reactive to Rhus suggest some basic difference in reaction
The inflammatory responses seen in windows of Rhus-negative subjects from 24 to 60 hours are
eosinophilie response showed evidence of bacin the windows from subjects having contact terial contamination.
pattern between allergic and non-allergic individuals. However, it should be noted that not all of our Rhus-positive subjects exhibited this eosinophilic response. The majority of these Rhus-positive subjects exhibiting atypical response had only weakly positive patch test to Rhus antigen (Table I). It appears reasonable to assume that a weak patch test reaction represents a lesser degree of sensitization.
An additional finding in the windows taken from Rhus-positive individuals exhibiting atypical inflammatory reaction (lack of eosinophils) was the invariable presence of bacterial contami-
represented in Figure 9. These inflammatory curves are characterized by repeated waves of large numbers of neutrophils. Between the waves
of neutrophils there appears to be an influx of maerophages. The percentage of the lymphocytes remains more or less constant. It should be noted that this response differs in some cases from the
atypical response seen in Rhus-positive individuals. Our findings in Rhus-positive, atopie individuals were indeed interesting. Preparations taken
from these patients did not demonstrate the eosinophilia present in our strongly Rhus-posi-
tive, non-atopie subjects and therefore the inflammatory curves are somewhat similar to our Rhus-negative, non-atopie individuals. Evidence of bacterial contamination was present in all but
two of our atopie subjects. The large multinucleated "epithelioid" giant cells were also absent.
It is not known whether the absence of the eosinophilic response in our atopic subjects is another example of the atopic "paradox" or due to bacterial contamination. The significance of the large multinucleated "epithelioid" giant cells which appeared during the later hours (from 48 to 60 hours) only in the Rhus-positive subjects is not known. BraunFia. 20. A mononuclear cell with ingested bac- steiner et al (2) who performed window experiteria. This probably represents a lymphocyte ments on patients utilizing tularin or tuberculin which exhibits phagoeytic activity. TABLE II Patch test and skin window results in atopic individuals Subject
Diagnosis
Patcb Test to Rbus
1
Atopie dermatitis* Atopic dermatitis Atopie dermatitis
Weakly positivet Weakly positive Positive Positive Positive Positive Negative Negative
2 3
4 5 6
Atopie
7
Atopie Atopie
8
Atopie
Atopie
Typical
Response
* Classical atopic dermatitis along with other evidence of atopy.
t Positive only at llhus extract 1:200 but negative to 1:1000. t Atopie stigmata but without classical atopie dermatitis.
Atypical Response
nactecia
x
x
X X X X X X
X X X
X
><
1< ><
HUMAN SKIN WINDOW
417
as antigen found massive infiltration of lympho- tionship between the presence of eosinophils and cytes which rapidly hypertrophied and became immune responses. In further studies be (8) con"mononuclear", "epithelioid", and even giant cluded that the eosinophilic response to injections cells. These derivatives of lymphocytes were of antigens paralleled antibody production. found to be capable of transferring delayed skin Torre and Leikin (9) performed window tests allergy in a highly specific manner. In this study, on patients with eosinopbilia. In their experithe authors found that the eosinophils were ments there were large numbers of eosinophils rather scarce in the exudate even in the presence during the early hours of the experiment. The of blood eosinophilia. Rebuck and Yates (3) also eosinopbils gradually decreased in number in noted the presence of large macrophages and subsequent windows but never completely disgiant cells in the tuberculin-positive windows at appeared. 63 hours. Riis (10) found no regular correlation between Rebuck and Yates (3) studied the cellular re- tbe number of eosinophilic granuloeytes in the sponses in windows in tuberculin-negative and peripheral blood and in the inflammatory exupositive subjects, employing Old Tuberculin as date. antigen in these studies. The inflammatory reCONCLU5ION5 sponses in subjects with negative tuberculin tests were similar to the responses to other nonpyo1. The skin window offers a dynamic approach genie antigens, i.e., diphtheria toxoid. Marked for studying the inflammatory exudate produced alterations in the neutrophilie sequences occurred by various types of antigens, irritants and in the tuberculin-positive subjects. These altera- trauma. The application of diphtheria toxoid tions resulted in defective and retarded mono- under the skin window produces inflammatory nuclear responses which were primarily a reflec- reaction of a nonpyogenic nature and serves as a tion of lymphocytic depression in the inflamma- proteotype or baseline for comparing other types tory cycle. The importance of a normal neutro- of stimulation. 2. Within the first 24 hours a skin window, philic response was further confirmed by Page and Good (4, 5) in an extensive study of inflam- utilizing diphtheria toxoid as the test substance, mation in a patient with cyclic neutropenia and will show early migration of pbagoeytic granuloin rabbits with experimentally induced neutro- cytes which are later supplemented by migrating
penia. They demonstrated that the absence of lymphocytes and blood monunuelears. In still neutrophils from the circulation profoundly later stages these mononuclear cells have been alters the early events of the inflammatiory cycle. Our experiment appears to illustrate the basic difference between tuberculin and contact ecze-
matous sensitivity. The lack of significant difference in the first 24-hour windows of Rhuspositive and Rhus-negative subjects is in direct contrast with what occurs in tuberculin allergy. The characteristic reaction patterns in the Rhuspositive windows did not become evident until
shown to hypertrophy to form additional macrophages to supplement tissue maerophages. 3. When Rhus oleoresin was applied under a skin window no difference in inflammatory pat-
tern between individuals positive by patch test to Rhus and individuals negative by patch test to Rhus was evident until the 33 hour stage. The Rhus-positive, aon-atopie individuals differed from the negative reactors as follows: (a) Eosino-
33 hours.
philia, (b) larger number of lymphocytes, and Eitzman and Smith (6), employing the skin (c) the presence of large multinueleated giant
window technic, described eosinophilic response cells. in newborn infants from two to twenty-one days 4. Bacterial contamination appeared to modify after delivery. The eosinophils appeared in the inflammatory pattern in the skin windows. windows from two hours to twelve hours. There 5. Atopic subjects appeared to react somewhat was no consistent high blood eosinophilia in these subjects.
differently from non-atopic, "normal" individuals.
Speirs (7) noted the accumulation of eosinoACKNOWLEDGMENTS phils between the second and the tenth day after The authors gratefully acknowledge the advice repeated injections of antigenie materials. His experiments suggested the existence of the rela- and assistance from Dr. J. W. Rebuck of the
418
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Department of Laboratories in the early phase of this study. REFERENCES 1. REBUCK, J. W. AND CROWLEY, J. H.: A method
experimental study of the function of neu-
trophils in the inflammatory response.
Amer. J. Path., 34: 645—669, 1958. 6. EITZMAN, D. V. AND SMITH, R. T.: The non-
specific inflammatory cycle in the neonated infant. A.M.A. J. Dis. Child., 97: 326—334, 1959.
of studying leukocytic functions in vivo.
7. SI'EIRS, R. S.: Physiological approaches to an
2. BRAUN5TEINER, H., PAERTAN, J. AND THUMB,
phils and basophils. Ann. N. Y. Acad. Sci.,
Blood, 13: 417—426, 1958. 3. REBUCIC, J. W. AND YATES, J. L.: The cytology
of the tuberculin reaction in skin windows
8. SPEIE5, R. S.: Advances in the knowledge of the eosinophil in relation to antibody formation. Ann. N. Y. Acad. Sci., 73: 283—306,
226, 1954.
9. TOREE, C. M. AND LEIKIN, S. L.: Tissue in-
Aon. N. Y. Acad. Sci., 59: 757—805, 1955.
N.: Studies on lymphocytic functions. in man. Amer. Rev. Tuberculosis, 69: 216—
4.
PAGE, A. R. AND GooD, R. A.: Studies on cyclic neutropenia. A.M.A. J. Dis. Child., 94: 623—661, 1957.
5. PAGE, A. R. AND Gooo, R. A.: A clinical and
understanding of the function of eosino59: 705—731, 1955.
1958.
flammatory cytology in clinical states. Amer. J. Chn. Path., 32: 335—345, 1959. 10. Rt's, P.: The Cytology of Inflammatory Exudate. Copenhagen. Munksgaard, 1959.
DISCUSSION Dn. ARTHUR W. GRACE (New York, New York): I was rather uncertain about the column headed "Bacteria" in Tables No. 1 and No. 2. It wasn't clear to me whether we are dealing with blister fluids in there or not. It has been my experience with spontaneously appearing blister fluids or those that were induced by the use of
to identify the bacteria in the windows. We
blister fluids remain bacteriologically sterile for about twenty-four to thirty-six hours after their formation. At what time after the appearance of such blisters as there were in these two tables, was the study for bacteria made known? Were the bacteria identified only by direct observation through a window or by direct observation and by staining?
as 12 hours after the initiation of the window. Employing the May-Gruenwald-Giemsa stain, the bacteria can be seen to increase in number from hour to hour in the phagocytic cells. There is no evidence of clinical infection even though the windows show bacterial contamination microscopically. In the Rhus-sensitive individuals, the denuded sites actually heal faster than the
DR. ROBERT W. GOLTI (Minneapolis, Minne-
intact skin around it. The unabraded skin around
believe that these organisms are normal resident as well as transient skin flora. Since the window sites arc sterilized only by superficial cleansing with alcohol before scraping, it would be expected
that some of the bacteria of the skin left behind
in the follicles eventually may move into the cantharides paste on normal skin, that such inflammatory site. We see these bacteria as early
sota): Dr. Fa,saro and Dr. Good of the Uni- the window eventually shows symptoms and versity of Mii;aesota have carried out this technic
over the past year or two. Their particular interest centered around dermatitis herpetiformis; and perhaps I shouldn't quote their results without consulting with them but, as I remember it, they showed an increase in cosinophil infiltration in the very early stages within the first twentyfour hours, in contrast to Dr. Punan Ru's findings.
signs of a classical contact dermatitis to Rhus. In response to Dr. Goltz's question and comments on dermatitis herpetiformis, we have done
skin windows in this condition, but we are not as yet ready to report our findings. We do see eosinophils appearing in the windows in the early
stages when the patients have a high blood eosinophil count. We consider this a nonspecific response. There are several reports of the cosino-
four hours, however, feeling that by that time
philic response in windows. The correlation between blood eosinophilia and tissue eosino-
bacterial contamination had become such a factor
philia vary so much that most observers consider
They gave up their observations after twenty-
that the observations were no longer reliable it non-significant. A high blood cosinophilia after that time. Perhaps they should have gone apparently may or may not be accompanied by on further. a high cosinophilic response in the windows. DR. FUNAN Ru (in closing): With regard to In all our experimental subjects, we do a difDr. Grace's question, we have made no attempt ferential white blood cell count at the beginning
HUMAN SKIN WINDOW
419
and at the end of 48 hours of the window experi- hers, and reach a peak from 48 to 55 hours. In ment. None of them shows any significant change contrast to the appearance of cosinophils in the of blood picture in the two specimens. first 24 hours which we consider as a non-specific In the windows of the Rhus-positive indi- response, the eosinophilic response in the Rhusviduals, the eosinophils can go up to as high as positive windows at the late hours, probably is 40 per cent. The eosinophils begin to appear at significant. This response is not found in persons 30 to 33 hour stage, gradually increase in num- with negative patch test to Rhus.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.