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The skin window test in rhinoscleroma
The Skin W i n d o w Test in R h i n o s c l e r o m a I-[USSEIN H. TOPPOZADA, D.L.O., M.S., F.A.C.S., F.I.C.S.,* L, MICHAELES, H. MAZLOUM, M. EL-SNWY, R. MALATY, AND Y. YAKOUT
The skin window test was applied to evaluate the state of immunity in patients with rhinoscleroma. The use of the patient's own Klebsiella micro-organisms as antigen led to a negligible change in the percentage of lymphoblasts, whereas the use of foreign Klebsiella micro-organisms as antigen resulted in a pronounced change, This finding indicates an impaired cellular immune response, Application of Klebsiella microorganisms as antigen to normal subjects led to a comparatively high percentage of lymphoblastic transformation. This finding suggests the possible use of a standard Klebsiella antigen as a vaccine. The possible use of the skin window test to identify people with deficient T lymphocyte function is discussed as well as its use in monitoring the efficacy of such a vaccine in influencing the cellular immune response.
Rhinoscleroma is a chronic granulomatous disease of the nose and upper respiratory system. Endemic loci exist in 25 countries in Europe, Asia, Africa, and America. 7 Clinically the disease has b e e n described in progressive stages: catarrhal, atrophic, granulomatous, and fibrotic. However, overlapping of the stages occurs in the individual patient# The histologic picture in rhinoscleroma is considered to he diagnostic and indicates that important i m m u n o l o g i c changes take place in this disease. There is an excess of plasma cells and Russell bodies, w h i c h may suggest marked B l y m p h o c y t e activity. On the other hand, the Mikulicz cells with their undigested Klebsiella
micro-organisms suggest that T lymphocyte function is reduced. The skin window test was first described by Roebuck and Crowley 'J to study the local inflammatory reactions in normal subjects and in diseased patients. The origin and characteristics of the macrophages in such prepm'ations have been studied b y numerous investigators. I~ 12.15. ic~The skin window test has been used to study the cellular inflammatory response in a variety of hematologic conditions. 6 The application of anti-IgD and performance of the skin window test in patients suffering from allergic contact dermatitis were found to induce an appreciable proliferation of lymphocytes, which migrated
Accepted for publication November 5, 1980, *Professor, E.N.T. Department, Alexandria Faculty of Medicine, Alexandria, Egypt.
30
Americml Journal of OtolaryngMegy --Volume 2, Number 1, Februmy 1981
onto the coverslip, a Cancanavallin A, a nonspecific T lymphocyte mitogen, reproduced the same phenomenon and seems to be a mitogon specific for T suppressor lymphocytes. ~" ~3,,T The purpose of our study was to investigate the value of the skin window test as a screening test for the macrophages and skin cellular kinetics in patients with rhinoscleroma and to assess its possible use for the evaluation of the immunologic status in such patients.
MATERIAL AND METHODS The study population included 30 patients with rhinoscleroma, of whom 16 were untreated (for tln'ee to seven years) and 14 were treated (specific antibiotics and irradiation), and 10 normal subjects. All patients were examined clinically, pathologically, and bacteriologically to confirm the diagnosis of rhinoscleroma. Biopsy material was taken from the nasal lesion and subjected to the following examinations. 1. ttistopathologic examination using paraffin sections and hematoxylin and eosin staining to show the characteristic picblre of rhinosclero-
tify specific antibodies. The sera in all patients with rhinoscleroma in this study showed a high titer, ranging from 1:160 to i:1280. ]'he skin window test was performed in all subjects. The bacterial antigen was prepared from a pure colony of Klebsiella type 3 in saline suspension, which was rapidly frozen and thawed five times. On the forearm three abrasions were made using a fine abrasive disc. The test substance was applied to a coverslip and inverted quickly and placed over the abrasion. To the first abrasion, saline was applied as a control, to the second the patient's own Klebsiella micro-organisms, and to the third foreign Klebsiella micro-organisms of the same seretype. The three coverslips were protected with silicon rubber and covered with gauze. The coverslips were removed and replaced with new coverslips every 24 hours 24, 48, and 72 hours after applications of the test substance, The cellular exudate thus obtained on the coverslip was stained wit1 Leishman's stain, and the percentages of the cellular populations were determined. D Alpha-naphthyl acetate esterase stain, which was helpful in cellular differentiation, was used in some cases (Fig. 1}. 1
Volume 2 Number 1
February 1981
ma.
2. Homogenization of tissue, which was inoculated on blood agar and MacConkey's agar. Pure colonies were selected and identified biochemically using the API 20 E system. 14 The agglutination test was done to identify the type of the micro-organism (all were Klebsiella type 3). In addition serum was collected, and the complement fixation test was performed to iden-
RESULTS Untreated Patients with Rhinoscleroma SALINE CONTROL. There was an immediate neutrophilic response followed by a monocytic response to the saline control. The responses were equal after 48 hours (Fig. 2). After 72 hours
Figure 1. Nonspecific esterase stain with alpha-naphfllylacetate esterase for monocytes.
HUSSEIN H. TOPPOZADA ET AL.
3]
American Journal
~80 ~70
%
of Otolaryngology
TABLE 1. Mean Percentages and One Standard Deviation of the Cellular R e s p o n s e s in the Skin W i n d o w Test to Saline in 30 Patients with Rhinoscleroma
->%-
50 (D "40 o ~o3 0
TIME A F T E R
TYPESOFCELLS
~20 ~I0
0
.
_
_
1
2 4 hr.
1
J
4 8 hr.
- / 2 hr.
TIME Figure 2. Response to saline in untreated patients with rhinoscleroma in the skin window test. Neutrophils ---. Monocy'tes. . . . Lymphob/asts there was degeneration of the cells with increasing numbers of s q u a m o u s epithelial cells and early infection, which obviated carrying the test further. Lymphoblasts could not be detected at any time, PATIENT'S OWN KLEBSIELLA MICRO-ORGANISMS AS ANTIGEN. Similar percentages of neutrophils and of monocytes were observed in response to the patient's own Klebsiella micro-organism as antigen and in response to saline (Tables i, 2). About 3 per cent of the cell p o p u l a t i o n appeared as large cells with a small a m o u n t of basophilic cytoplasm around large, deeply stained nuclei with finely granular chromatin a n d an occasional nucleolus (Fig. 3]. The percentage of l y m p h o cytes r e m a i n e d ' t h e same at each time of examination (Fig. 4). The appearance of l y m p h o c y t e s was considered to represent l y m p h o b l a s t i c transformation, a manifestation of cell m e d i a t e d i m m u n i t y to Klebsiella infection. FOREIGN KLEBSIELLA M~CRO-ORGANISMS ANTIGEN. The f o r e i g n K]ebsie]]a m i c r o - o r g a n i s m s used as antigen p r o d u c e d the same neutrophilic and monocytic response as did the patient's o w n Klebsiella micro-organisms (Tables 2, 3). How-
24 Hr.
Eosinophils Neutrophils Lymphocytes Lyraphabtasts Monocytes
2.3 62.2 14.4 0,1 20.3
• 2.3 ~ 5.1 -+ 4.9 • 0,3 4- 5.2
APPLICATION
48 Hr.
4.2 4- 3.2 40.9 -- 7.3 17.5 -+-+6.8 0.9 • 0.7 36.3 • 5.5
72 Hr.
4.1 -+ 2 20.5 4-- 7.6 19.1 -- 7,4 1.O 1.2 55.1 4- 9.5 -
ever, the l y m p h o b l a s t s r e p r e s e n t e d a b o u t 13 per cent of the cell p o p u l a t i o n (Fig, 5). This response (13 per cent) is significantly different (P = 0.01) from the 3 per cent r e s p o n s e of u n t r e a t e d patients to their o w n Klebsiella m i c r o - o r g a n i s m . The cellular r e s p o n s e did n a t differ a m o n g the patients in the various c l i n i c o p a t h o l o g i c stages of the disease,
Normal Subjects SALINE CONTROL. The response to saline was the same in normal subjects as it was in untreated patients, FRESH KLEBSIELLA MICRO-ORGANISMS AS ANTIGEN. The lymphoblastic response to fresh Klebsiella m i c r o - o r g a n i s m s as a n t i g e n in normal subjects was a p p r o x i m a t e l y 31 per cent of the cell p o p u l a t i o n (Figs. 6, 7; Table 4). This r e s p o n s e (31 per cent) is s i g n i f i c a n t l y different (P = 0.01) from the 3 p e r c e n t r e s p o n s e of u n t r e a t e d patients to their o w n Klebsiella microo r g a n i s m and from the 13 per c e n t r e s p o n s e of u n t r e a t e d patients to f o r e i g n Klebsiella microorganisms.
Treated Patients with Rhinoscleroma The l y m p h o b l a s t i c response i n treated patients with r h i n o s c l e r o m a did n o t differ from
TABLE 2. Mean Percentages and One Standard Deviation of the Cellular Responses in the Skin W i n d o w Test in 30 Patients with Rhinoscleroma to the Patient's Own Klebsiella Micro-organism as A n t i g e n
TYPESOF CELLS
TIMEAFTERAPPLICATION 24 Hr, 48 Hr. Total Response Specific Total Response Specific to Saline Responseto to Saline Responseto and Antigen Antigen and Antigen Antigen
72 Hr. Total Response Specific to Saline Responseto and Antigen Antigen
Eosinophils Neutrophils
4,2 • 3,4 59.8 • 9.9
1.9% -2.4%
5.1 +- 4.4 37.2 - 6.7
0.9% -3.7%
4.7 4- 5.4 20.4 -+ 6.2
0.6% -0.13%
Lymphocytes
13.6 +- 6.2
-0.6%
15.9 +- 5.6
-1.6%
18.2 + 8.6
-0.9%
Lymphoblasts Monocytes
2.5 -~ 1,5 19.4 • 5.9
2.4%
3,3 ~ 1.7 37,7 ~- 5.7
2,4% 1.4%
3.6 ~ 1.5 52.9 + 9.5
2.6% --2.2%
--0.9%
N.B. The specificresponse to antigen is the difference between the response to saline (Table 1) and the response to saline and antigen, 32
THE SKIN WINDOW TEST IN RHINOSCLEROMA
Volume 2 Number 1 February 1981
Figure 3.
Predominantly manocyfic r e s p o n s e in a p a t i e n t with rhinos d a r o m a to t h e p a t i e n t ' s o w n Klebsiella micro-orgarfisms, Note the [ y m p h o b l a s t (arrow),
o ~-
8O 70 ,,.,.
~ 6O ~.50 "~ 4 0
Figure 4. R e s p o n s e to untreated patient's o w n Klebsiella m i c r o - o r g a n i s m s as antigen in the s k i n w i n d o w test. Neut r o p h f i s - - - , lvlonocytes . . . . L y m p h o b l a s t s _
r
, c~ ,.-30 ~20
s., ~. "~
O
2 4 hr. 4 8 hr. TIME
r T A B L E 3,
~
7'2 hr.
M e a n P e r c e n t a g e s a n d O n e S t a n d a r d D e v i a t i o n of the Cellular R e s p o n s e s in t h e S k / n W i n d o w Test i n 30 P a t i e n t s w i t h R h i n o s c ] e r o m o to F o r e i g n Klebsiella M i c r o - o r g a n i s m s a s A n t i g e n
24 Hr, TYPES OF CELLS
Eosinophils Neutrophila Lymphocytes Lymphob]asts Monocytes
Total R e s p o n s e to Saline a n d Antigen Meun % • S.D. 4,4 53,5 14.7 11.2 16.0
Specific
Response to Antigen
TIME AFTER APPLICATION 48 Hr, Total Response Specific
to Saline and Antigen
Response to Antigen
Mean % +- S,D,
• 3 • 10.4 • 4.8 ~ 2.6 • 5.8
2.1% -8.7% 0.3% 11.1% 4.3%
5.1 33.6 15.4 13.4 32.4
-+ 3.3 --- 4.9 • 6.1 • 3.2 • 5.7
0,9% -7.3% -2.1% 12.5% 4.0%
72 Hr. Total Response Specific to Saline Response to and Antigen Antigen Mean % +- S.D. 5,3 15.7 13.0 15.0 50.9
• • -
4.1 6.7 5.3 3.2 8.2
1.2% -4.8% -6,1% 14% -4.2%
N,B. T h e s p e c i f i c r e s p o n s e to a z t i g e n :~s tim difference between t h e r e s p o n s e to saline (Table 1) and t h e r e s p o n s e to saline a n d
antigen. T A B L E 4.
M e a n P e r c e n t a g e s a n d O n e S t a n d a r d D e v i a t i o n of the Cellular R e s p o n s e s in the Skin W i n d o w Test in 10 N o r m a l S u b j e c t s to Klebsiella M i c r a - o r g a n i s m s as A n t i g e n TiME APrER APPLICATION 48 Hr.
24 Fir.
TYPES OF CELLS
Eosinophils Neutrophils Lymphocytes Lymphoblasts Monocytes
Total to and Mean
Response Saline Antigen
Specific Response to Antigen
% • S.D.
0.2 47.0 6.0 39.0 16.8
• • • • •
0.4 8.4 4.8 6.9 3.8
- 2.1% -15.2% - 8.4% 29.9% -3.5%
Total Response
Specific
to Saline
R e s p o n s e to
and A n t i g e n Mean % _-t S.D.
Antigen
2.6 22.6 9.4 29.8 35.6
-+ 1.7 - 3 • 3.3 -- 8.3 • 9.2
-1.6% -18.3% -8.1% 28.9% -0.7%
72 Hr. Total Response Specific to Saline and A n t i g e n Mean % ~ S.D.
Response to Antigen
0.2 • 0.4 13.3 • 4.8
-3.9% -7.2%
0.4 • 0.7
-18.7%
33.4 +- 8.4 53.2 9 6.8
32.4% -1.9%
N.B. T h e s p e c i f i c r e s p o n s e to a n t i g e n is the difference between the response to salino (Table 1} and the responsa to saline a n d antigen.
33
American Journa| of Otolaryngology
~
eO 7O
~
60
~80 "-" 7 0 o
,s
~ 5O
~ 40 j,~e
~o ~ 0
~,2o
tr
~
"~
~
20
-I2:~ 1 0
I
2 4 hr. 4 8 hr. TIME
7 2 hr.
U
ck
0
....
I
2 4 hr. 4 8 hr. TIME
7 2 hr.
Figure 5. R e s p o n s e to foreign Klehsiella m i c r o - o r g a n i s m s as antigen in u n t r e a t e d patients w i t h r h i n o s c l e r o m a in the skin w i n d o w test. N e u t r e p h i l s - - - . M o n o c y l e s . . . . Lymphoblasts ~ .
F i g u r e 6. R e s p o n s e to Klehsiella m i c r o - o r g a n i s m s as a n t i g e n in n o r m s [ subjects in t h e s l d R w i n d o w test. N e u t r o p h i l s - - - . Monocytes .... Lymphoblasts ~ .
that in untreated patients with rhinoscIeroma to the saline, the patient's own Klebsiella microorganisms as antigen, and foreign Klebss micro-organisms as antigen.
capsular antigen in the stxains encountered m a y be an alternative explanation. The possibility that the antigenicity of the patient's o w n microorganisms has become attenuated exists and may be of importance. Against such a h y p o t h e s i s is the potent lymphoblastic transformation encountered in normal control subjects in w h o m the same micro-organism was used. There was a high titer in the c o m p l e m e n t fixation test in all the patients with rhinoscleroma, which is indicative of the presence of specific antibodies for the patient's Klebsiella antigen. The following questions arise: Does this observed change in lymphocytic transformation represent a specific defect in T l y m p h o c y t e function in such patients, or are such effects nonspeel fie and due to the lesion or general condition of the patient? The percentage of lymphoblasts did n o t differ
DISCUSSION The use of the patient's own Klebsiella microorganism as antigen produced a lymphoblastic transformation in the skin w i n d o w test. However, the use of a foreign Klebsiella microorganism as antigen in patients with rhinoscleroma resulted in a more pronounced effect. These findings may be indicative of a state of tolerance to the already present Klebsiella antigen. The variations encountered in the O antigen despite the presence of a uniform type 3
F i g u r e 7. L y m p h o b l a s t i c r e s p o n s e in a n o r m a l s u b j e c t to K l e b s i e l l a m i c r o o r g a n i s m as a n t i g e n .
34
T H E SKIN WINI3OW TEST IN R H I N O S C I , E R O M A
in the treated and the untreated patients. Furthermore, the nature of the lesion had no direct impact on the lympheblastic response, for patients with active granuloma formation showed almost the same response as those with lesions in the fibrotic stage. These observations regarding the cellular response may suggest that patients with depressed T lymphocyte function are those who are more likely to contract this Klebsiella infection. This concept may be substantiated by the clinical observation that there is a variable incidence of the disease in members of the same family who m'e exposed to the same source of infection. It appears that patients with rhinoscleroma are characterized by impairment of T lymphocyte function and by concomitantly stimulated antibody formation (humeral immunity). Rhinoscleroma could be considered as an acquired immunodeficiency disease in which there is a progressive failure of the immunologic responsiveness, which might result in worsening or progression of the disease in spite of treatment. ~ Such a paradoxical response was f o u n d to o c c u r in s o m e
endemic
parasitic
dis-
eases in Egypt such as schistosomiasis. '~ Zakrzewski ~s investigated the etiopathology of rhinoscleroma, and it was found that there must coexist another exogenous or endogenous factor, because the infective agent per se does not account for the development of the disease. The low nutritional state ef the affected population might be of significance in this respect. Serum protein, iron, and copper levels are low in patients with rhinescleroma and may be factors in the pathogenesis of the disease. Neumans found that in malnourished chihtren, the principal deficiencies are found in T lymphocytes and their responses, whereas the levels of all component fractions of the immunoglobulins are high and antibody production is normal. The skin window test using Klebsiella antigen may allow us to identify contacts with depressed T lymphocyte function who might be susceptible to rhinoscleroma after prolonged contact with the disease. A vaccine for contacts may help to improve their immune status. The skin window test may be a useful tool in evaluating the efficacy of such a vaccine in influencing the cellular immune mechanisms of the host. The test is easy and inexpensive to apply.
O t o l a r y n g o l o g y , and Dr. N. S b a b a a n , P r o f e s s o r of Histology, A l e x a n d r i a University, for t h e i r coliaboraLion and h e l p in the p r e s e n t work.
Volume 2 Number 1 February 1981
References 1. Allison, A. C., Horwitz, D. H., Ward, P., and Kight, K.: Identification of human mononuclear leucocyte population by esterase staining. Clin. Exp. Immunol., 30:289, 1977. 2. Castro, ]. E.: Immunology for Surgeons. Mechanisms Underlying Immunological Diseases. MTP Press Ltd., 1976, pp. 60-90. 3. Dikeacou, T., and Cormane, R.: Study of the inflammation induced by IgD using skin window technique. Arch. Dermatol. Res., 1980. 4. Ei-Mofti, A., Imam, A., Bcutros, G., Hamilton, P., and Floyd, T.: Scleroma in Egypt. Ann. Otol. Rhin61. Laryngol., S3;1031-1056, 19{}4. 5. Ghanem, M,, Sadek, A., and El-Sawy, M.: Effects of splenectomy on serum immunoglobulins in schistosomal hepatic fibrosis. Ain Shams Med. J., 27:57, 1976. 6. Ctlosh, M. L., Hudson, G., and Blackburn, E. K.: Skin window macrophages in malignant lymphomas, Br. 1. Haematol., 25:293-297, 1973. 7. Muzyka, M. M., and Gubina, K. M.: Problems of the epidemiology of scleroma, I. Hyg. Epidemld. Microbiol. Immunol., 16:1, 1972. 8. Neumann, C. G., Lawlor, G. T. T., Stiehn, E. R., Swendseid, M. E,, Newton, C., Herbert, J., Ammann, A. J., and Jacobs, M.: Immunologic responses in malnourished children. Am, [. Nutr., 28 ',89, 1975. 9. Roebuck, J. W., and Crowley, J. H.: A method of studying leukouytic functions in rive, Ann. N. Y. Acad. Sci., 59:757, 1955. 10. Roebuck, J. W,, Monte, R. W., Monagham, E. A., and Riddle, J. M.: Potentialities of the lymphocyte with an additional reference to its dysfunction in Hodgkin's disease. Ar~n. N, Y. Acad, Sci., 73:8, 1958. 11. Russel, R. ]., .Wilkinson, P. C., Sless, F., and Parrot, O. M.: Chemotaxfs of lymphob[ast. Nature (Lend}., 256:646, 1975. 12. Schmalzl, F., Huber, H., Asamer, K., Abbrederis, K., and Braunsteiner, H.: Cytochemical and imnmnohistologic investigations on the source and the functional changes of mononuclear cells in skin window exudates. Blood, 34:129, 1969. 13. Schreiner, G. F., and Unanue, E. R.: Anfi-Ig triggered movements of lymphocytes; specificity and lack of evidence for directional migration. J. Immunol., 114:809, 1975. 14. Smith, P. B., Tomfohrde, K. M., Rhoden, D. L., and Balows, A.: APt system, a multitube micromethod for identification of Enterobacteriaceae. Appl. Microbiol., 24:449-452, 1972. 15. Trepel, F., end Begemann, H.: On the origin of the skin window macrophages. Aeta Haomato[., 36:386, 1966. 16. Van Furth, R.'. Origin and kinetics of monocytes end macrophages. Semin. Hamnatol., 7:125, 1970. 17. Wilkinson, P. C., Roberts, J. A., Russel, R. J., and McLoughlin, M.: Chemotaxis of mitogen-activated human lymphocytes and the effect of membrane active enzymes. Clin. Exp. Immuno[., 25:289, 1976. 18. Zakrzewski, A.: On the importance of trace elements for mucosa of upper air passages. Acta Otolaryngol., 65:55-58, 1968.
Acknowledgment The authors are grateful to Mr. I. Boxall, Principal Scientific Laboratory Officer of the L o n d o n Institute of
HUSSEIN H. TOPPOZADA ET AL.
19 Midan Sahad Zaghloul Alexandria Egypt (Dr. Toppozada)
35
The skin window test was applied to evaluate the state of immunity in patients with rhinoscleroma. The use of the patient's own Klebsiella micro-organisms as antigen led to a negligible change in the percentage of lymphoblasts, whereas the use of foreign Klebsiella micro-organisms as antigen resulted in a pronounced change, This finding indicates an impaired cellular immune response, Application of Klebsiella microorganisms as antigen to normal subjects led to a comparatively high percentage of lymphoblastic transformation. This finding suggests the possible use of a standard Klebsiella antigen as a vaccine. The possible use of the skin window test to identify people with deficient T lymphocyte function is discussed as well as its use in monitoring the efficacy of such a vaccine in influencing the cellular immune response.
Rhinoscleroma is a chronic granulomatous disease of the nose and upper respiratory system. Endemic loci exist in 25 countries in Europe, Asia, Africa, and America. 7 Clinically the disease has b e e n described in progressive stages: catarrhal, atrophic, granulomatous, and fibrotic. However, overlapping of the stages occurs in the individual patient# The histologic picture in rhinoscleroma is considered to he diagnostic and indicates that important i m m u n o l o g i c changes take place in this disease. There is an excess of plasma cells and Russell bodies, w h i c h may suggest marked B l y m p h o c y t e activity. On the other hand, the Mikulicz cells with their undigested Klebsiella
micro-organisms suggest that T lymphocyte function is reduced. The skin window test was first described by Roebuck and Crowley 'J to study the local inflammatory reactions in normal subjects and in diseased patients. The origin and characteristics of the macrophages in such prepm'ations have been studied b y numerous investigators. I~ 12.15. ic~The skin window test has been used to study the cellular inflammatory response in a variety of hematologic conditions. 6 The application of anti-IgD and performance of the skin window test in patients suffering from allergic contact dermatitis were found to induce an appreciable proliferation of lymphocytes, which migrated
Accepted for publication November 5, 1980, *Professor, E.N.T. Department, Alexandria Faculty of Medicine, Alexandria, Egypt.
30
Americml Journal of OtolaryngMegy --Volume 2, Number 1, Februmy 1981
onto the coverslip, a Cancanavallin A, a nonspecific T lymphocyte mitogen, reproduced the same phenomenon and seems to be a mitogon specific for T suppressor lymphocytes. ~" ~3,,T The purpose of our study was to investigate the value of the skin window test as a screening test for the macrophages and skin cellular kinetics in patients with rhinoscleroma and to assess its possible use for the evaluation of the immunologic status in such patients.
MATERIAL AND METHODS The study population included 30 patients with rhinoscleroma, of whom 16 were untreated (for tln'ee to seven years) and 14 were treated (specific antibiotics and irradiation), and 10 normal subjects. All patients were examined clinically, pathologically, and bacteriologically to confirm the diagnosis of rhinoscleroma. Biopsy material was taken from the nasal lesion and subjected to the following examinations. 1. ttistopathologic examination using paraffin sections and hematoxylin and eosin staining to show the characteristic picblre of rhinosclero-
tify specific antibodies. The sera in all patients with rhinoscleroma in this study showed a high titer, ranging from 1:160 to i:1280. ]'he skin window test was performed in all subjects. The bacterial antigen was prepared from a pure colony of Klebsiella type 3 in saline suspension, which was rapidly frozen and thawed five times. On the forearm three abrasions were made using a fine abrasive disc. The test substance was applied to a coverslip and inverted quickly and placed over the abrasion. To the first abrasion, saline was applied as a control, to the second the patient's own Klebsiella micro-organisms, and to the third foreign Klebsiella micro-organisms of the same seretype. The three coverslips were protected with silicon rubber and covered with gauze. The coverslips were removed and replaced with new coverslips every 24 hours 24, 48, and 72 hours after applications of the test substance, The cellular exudate thus obtained on the coverslip was stained wit1 Leishman's stain, and the percentages of the cellular populations were determined. D Alpha-naphthyl acetate esterase stain, which was helpful in cellular differentiation, was used in some cases (Fig. 1}. 1
Volume 2 Number 1
February 1981
ma.
2. Homogenization of tissue, which was inoculated on blood agar and MacConkey's agar. Pure colonies were selected and identified biochemically using the API 20 E system. 14 The agglutination test was done to identify the type of the micro-organism (all were Klebsiella type 3). In addition serum was collected, and the complement fixation test was performed to iden-
RESULTS Untreated Patients with Rhinoscleroma SALINE CONTROL. There was an immediate neutrophilic response followed by a monocytic response to the saline control. The responses were equal after 48 hours (Fig. 2). After 72 hours
Figure 1. Nonspecific esterase stain with alpha-naphfllylacetate esterase for monocytes.
HUSSEIN H. TOPPOZADA ET AL.
3]
American Journal
~80 ~70
%
of Otolaryngology
TABLE 1. Mean Percentages and One Standard Deviation of the Cellular R e s p o n s e s in the Skin W i n d o w Test to Saline in 30 Patients with Rhinoscleroma
->%-
50 (D "40 o ~o3 0
TIME A F T E R
TYPESOFCELLS
~20 ~I0
0
.
_
_
1
2 4 hr.
1
J
4 8 hr.
- / 2 hr.
TIME Figure 2. Response to saline in untreated patients with rhinoscleroma in the skin window test. Neutrophils ---. Monocy'tes. . . . Lymphob/asts there was degeneration of the cells with increasing numbers of s q u a m o u s epithelial cells and early infection, which obviated carrying the test further. Lymphoblasts could not be detected at any time, PATIENT'S OWN KLEBSIELLA MICRO-ORGANISMS AS ANTIGEN. Similar percentages of neutrophils and of monocytes were observed in response to the patient's own Klebsiella micro-organism as antigen and in response to saline (Tables i, 2). About 3 per cent of the cell p o p u l a t i o n appeared as large cells with a small a m o u n t of basophilic cytoplasm around large, deeply stained nuclei with finely granular chromatin a n d an occasional nucleolus (Fig. 3]. The percentage of l y m p h o cytes r e m a i n e d ' t h e same at each time of examination (Fig. 4). The appearance of l y m p h o c y t e s was considered to represent l y m p h o b l a s t i c transformation, a manifestation of cell m e d i a t e d i m m u n i t y to Klebsiella infection. FOREIGN KLEBSIELLA M~CRO-ORGANISMS ANTIGEN. The f o r e i g n K]ebsie]]a m i c r o - o r g a n i s m s used as antigen p r o d u c e d the same neutrophilic and monocytic response as did the patient's o w n Klebsiella micro-organisms (Tables 2, 3). How-
24 Hr.
Eosinophils Neutrophils Lymphocytes Lyraphabtasts Monocytes
2.3 62.2 14.4 0,1 20.3
• 2.3 ~ 5.1 -+ 4.9 • 0,3 4- 5.2
APPLICATION
48 Hr.
4.2 4- 3.2 40.9 -- 7.3 17.5 -+-+6.8 0.9 • 0.7 36.3 • 5.5
72 Hr.
4.1 -+ 2 20.5 4-- 7.6 19.1 -- 7,4 1.O 1.2 55.1 4- 9.5 -
ever, the l y m p h o b l a s t s r e p r e s e n t e d a b o u t 13 per cent of the cell p o p u l a t i o n (Fig, 5). This response (13 per cent) is significantly different (P = 0.01) from the 3 per cent r e s p o n s e of u n t r e a t e d patients to their o w n Klebsiella m i c r o - o r g a n i s m . The cellular r e s p o n s e did n a t differ a m o n g the patients in the various c l i n i c o p a t h o l o g i c stages of the disease,
Normal Subjects SALINE CONTROL. The response to saline was the same in normal subjects as it was in untreated patients, FRESH KLEBSIELLA MICRO-ORGANISMS AS ANTIGEN. The lymphoblastic response to fresh Klebsiella m i c r o - o r g a n i s m s as a n t i g e n in normal subjects was a p p r o x i m a t e l y 31 per cent of the cell p o p u l a t i o n (Figs. 6, 7; Table 4). This r e s p o n s e (31 per cent) is s i g n i f i c a n t l y different (P = 0.01) from the 3 p e r c e n t r e s p o n s e of u n t r e a t e d patients to their o w n Klebsiella microo r g a n i s m and from the 13 per c e n t r e s p o n s e of u n t r e a t e d patients to f o r e i g n Klebsiella microorganisms.
Treated Patients with Rhinoscleroma The l y m p h o b l a s t i c response i n treated patients with r h i n o s c l e r o m a did n o t differ from
TABLE 2. Mean Percentages and One Standard Deviation of the Cellular Responses in the Skin W i n d o w Test in 30 Patients with Rhinoscleroma to the Patient's Own Klebsiella Micro-organism as A n t i g e n
TYPESOF CELLS
TIMEAFTERAPPLICATION 24 Hr, 48 Hr. Total Response Specific Total Response Specific to Saline Responseto to Saline Responseto and Antigen Antigen and Antigen Antigen
72 Hr. Total Response Specific to Saline Responseto and Antigen Antigen
Eosinophils Neutrophils
4,2 • 3,4 59.8 • 9.9
1.9% -2.4%
5.1 +- 4.4 37.2 - 6.7
0.9% -3.7%
4.7 4- 5.4 20.4 -+ 6.2
0.6% -0.13%
Lymphocytes
13.6 +- 6.2
-0.6%
15.9 +- 5.6
-1.6%
18.2 + 8.6
-0.9%
Lymphoblasts Monocytes
2.5 -~ 1,5 19.4 • 5.9
2.4%
3,3 ~ 1.7 37,7 ~- 5.7
2,4% 1.4%
3.6 ~ 1.5 52.9 + 9.5
2.6% --2.2%
--0.9%
N.B. The specificresponse to antigen is the difference between the response to saline (Table 1) and the response to saline and antigen, 32
THE SKIN WINDOW TEST IN RHINOSCLEROMA
Volume 2 Number 1 February 1981
Figure 3.
Predominantly manocyfic r e s p o n s e in a p a t i e n t with rhinos d a r o m a to t h e p a t i e n t ' s o w n Klebsiella micro-orgarfisms, Note the [ y m p h o b l a s t (arrow),
o ~-
8O 70 ,,.,.
~ 6O ~.50 "~ 4 0
Figure 4. R e s p o n s e to untreated patient's o w n Klebsiella m i c r o - o r g a n i s m s as antigen in the s k i n w i n d o w test. Neut r o p h f i s - - - , lvlonocytes . . . . L y m p h o b l a s t s _
r
, c~ ,.-30 ~20
s., ~. "~
O
2 4 hr. 4 8 hr. TIME
r T A B L E 3,
~
7'2 hr.
M e a n P e r c e n t a g e s a n d O n e S t a n d a r d D e v i a t i o n of the Cellular R e s p o n s e s in t h e S k / n W i n d o w Test i n 30 P a t i e n t s w i t h R h i n o s c ] e r o m o to F o r e i g n Klebsiella M i c r o - o r g a n i s m s a s A n t i g e n
24 Hr, TYPES OF CELLS
Eosinophils Neutrophila Lymphocytes Lymphob]asts Monocytes
Total R e s p o n s e to Saline a n d Antigen Meun % • S.D. 4,4 53,5 14.7 11.2 16.0
Specific
Response to Antigen
TIME AFTER APPLICATION 48 Hr, Total Response Specific
to Saline and Antigen
Response to Antigen
Mean % +- S,D,
• 3 • 10.4 • 4.8 ~ 2.6 • 5.8
2.1% -8.7% 0.3% 11.1% 4.3%
5.1 33.6 15.4 13.4 32.4
-+ 3.3 --- 4.9 • 6.1 • 3.2 • 5.7
0,9% -7.3% -2.1% 12.5% 4.0%
72 Hr. Total Response Specific to Saline Response to and Antigen Antigen Mean % +- S.D. 5,3 15.7 13.0 15.0 50.9
• • -
4.1 6.7 5.3 3.2 8.2
1.2% -4.8% -6,1% 14% -4.2%
N,B. T h e s p e c i f i c r e s p o n s e to a z t i g e n :~s tim difference between t h e r e s p o n s e to saline (Table 1) and t h e r e s p o n s e to saline a n d
antigen. T A B L E 4.
M e a n P e r c e n t a g e s a n d O n e S t a n d a r d D e v i a t i o n of the Cellular R e s p o n s e s in the Skin W i n d o w Test in 10 N o r m a l S u b j e c t s to Klebsiella M i c r a - o r g a n i s m s as A n t i g e n TiME APrER APPLICATION 48 Hr.
24 Fir.
TYPES OF CELLS
Eosinophils Neutrophils Lymphocytes Lymphoblasts Monocytes
Total to and Mean
Response Saline Antigen
Specific Response to Antigen
% • S.D.
0.2 47.0 6.0 39.0 16.8
• • • • •
0.4 8.4 4.8 6.9 3.8
- 2.1% -15.2% - 8.4% 29.9% -3.5%
Total Response
Specific
to Saline
R e s p o n s e to
and A n t i g e n Mean % _-t S.D.
Antigen
2.6 22.6 9.4 29.8 35.6
-+ 1.7 - 3 • 3.3 -- 8.3 • 9.2
-1.6% -18.3% -8.1% 28.9% -0.7%
72 Hr. Total Response Specific to Saline and A n t i g e n Mean % ~ S.D.
Response to Antigen
0.2 • 0.4 13.3 • 4.8
-3.9% -7.2%
0.4 • 0.7
-18.7%
33.4 +- 8.4 53.2 9 6.8
32.4% -1.9%
N.B. T h e s p e c i f i c r e s p o n s e to a n t i g e n is the difference between the response to salino (Table 1} and the responsa to saline a n d antigen.
33
American Journa| of Otolaryngology
~
eO 7O
~
60
~80 "-" 7 0 o
,s
~ 5O
~ 40 j,~e
~o ~ 0
~,2o
tr
~
"~
~
20
-I2:~ 1 0
I
2 4 hr. 4 8 hr. TIME
7 2 hr.
U
ck
0
....
I
2 4 hr. 4 8 hr. TIME
7 2 hr.
Figure 5. R e s p o n s e to foreign Klehsiella m i c r o - o r g a n i s m s as antigen in u n t r e a t e d patients w i t h r h i n o s c l e r o m a in the skin w i n d o w test. N e u t r e p h i l s - - - . M o n o c y l e s . . . . Lymphoblasts ~ .
F i g u r e 6. R e s p o n s e to Klehsiella m i c r o - o r g a n i s m s as a n t i g e n in n o r m s [ subjects in t h e s l d R w i n d o w test. N e u t r o p h i l s - - - . Monocytes .... Lymphoblasts ~ .
that in untreated patients with rhinoscIeroma to the saline, the patient's own Klebsiella microorganisms as antigen, and foreign Klebss micro-organisms as antigen.
capsular antigen in the stxains encountered m a y be an alternative explanation. The possibility that the antigenicity of the patient's o w n microorganisms has become attenuated exists and may be of importance. Against such a h y p o t h e s i s is the potent lymphoblastic transformation encountered in normal control subjects in w h o m the same micro-organism was used. There was a high titer in the c o m p l e m e n t fixation test in all the patients with rhinoscleroma, which is indicative of the presence of specific antibodies for the patient's Klebsiella antigen. The following questions arise: Does this observed change in lymphocytic transformation represent a specific defect in T l y m p h o c y t e function in such patients, or are such effects nonspeel fie and due to the lesion or general condition of the patient? The percentage of lymphoblasts did n o t differ
DISCUSSION The use of the patient's own Klebsiella microorganism as antigen produced a lymphoblastic transformation in the skin w i n d o w test. However, the use of a foreign Klebsiella microorganism as antigen in patients with rhinoscleroma resulted in a more pronounced effect. These findings may be indicative of a state of tolerance to the already present Klebsiella antigen. The variations encountered in the O antigen despite the presence of a uniform type 3
F i g u r e 7. L y m p h o b l a s t i c r e s p o n s e in a n o r m a l s u b j e c t to K l e b s i e l l a m i c r o o r g a n i s m as a n t i g e n .
34
T H E SKIN WINI3OW TEST IN R H I N O S C I , E R O M A
in the treated and the untreated patients. Furthermore, the nature of the lesion had no direct impact on the lympheblastic response, for patients with active granuloma formation showed almost the same response as those with lesions in the fibrotic stage. These observations regarding the cellular response may suggest that patients with depressed T lymphocyte function are those who are more likely to contract this Klebsiella infection. This concept may be substantiated by the clinical observation that there is a variable incidence of the disease in members of the same family who m'e exposed to the same source of infection. It appears that patients with rhinoscleroma are characterized by impairment of T lymphocyte function and by concomitantly stimulated antibody formation (humeral immunity). Rhinoscleroma could be considered as an acquired immunodeficiency disease in which there is a progressive failure of the immunologic responsiveness, which might result in worsening or progression of the disease in spite of treatment. ~ Such a paradoxical response was f o u n d to o c c u r in s o m e
endemic
parasitic
dis-
eases in Egypt such as schistosomiasis. '~ Zakrzewski ~s investigated the etiopathology of rhinoscleroma, and it was found that there must coexist another exogenous or endogenous factor, because the infective agent per se does not account for the development of the disease. The low nutritional state ef the affected population might be of significance in this respect. Serum protein, iron, and copper levels are low in patients with rhinescleroma and may be factors in the pathogenesis of the disease. Neumans found that in malnourished chihtren, the principal deficiencies are found in T lymphocytes and their responses, whereas the levels of all component fractions of the immunoglobulins are high and antibody production is normal. The skin window test using Klebsiella antigen may allow us to identify contacts with depressed T lymphocyte function who might be susceptible to rhinoscleroma after prolonged contact with the disease. A vaccine for contacts may help to improve their immune status. The skin window test may be a useful tool in evaluating the efficacy of such a vaccine in influencing the cellular immune mechanisms of the host. The test is easy and inexpensive to apply.
O t o l a r y n g o l o g y , and Dr. N. S b a b a a n , P r o f e s s o r of Histology, A l e x a n d r i a University, for t h e i r coliaboraLion and h e l p in the p r e s e n t work.
Volume 2 Number 1 February 1981
References 1. Allison, A. C., Horwitz, D. H., Ward, P., and Kight, K.: Identification of human mononuclear leucocyte population by esterase staining. Clin. Exp. Immunol., 30:289, 1977. 2. Castro, ]. E.: Immunology for Surgeons. Mechanisms Underlying Immunological Diseases. MTP Press Ltd., 1976, pp. 60-90. 3. Dikeacou, T., and Cormane, R.: Study of the inflammation induced by IgD using skin window technique. Arch. Dermatol. Res., 1980. 4. Ei-Mofti, A., Imam, A., Bcutros, G., Hamilton, P., and Floyd, T.: Scleroma in Egypt. Ann. Otol. Rhin61. Laryngol., S3;1031-1056, 19{}4. 5. Ghanem, M,, Sadek, A., and El-Sawy, M.: Effects of splenectomy on serum immunoglobulins in schistosomal hepatic fibrosis. Ain Shams Med. J., 27:57, 1976. 6. Ctlosh, M. L., Hudson, G., and Blackburn, E. K.: Skin window macrophages in malignant lymphomas, Br. 1. Haematol., 25:293-297, 1973. 7. Muzyka, M. M., and Gubina, K. M.: Problems of the epidemiology of scleroma, I. Hyg. Epidemld. Microbiol. Immunol., 16:1, 1972. 8. Neumann, C. G., Lawlor, G. T. T., Stiehn, E. R., Swendseid, M. E,, Newton, C., Herbert, J., Ammann, A. J., and Jacobs, M.: Immunologic responses in malnourished children. Am, [. Nutr., 28 ',89, 1975. 9. Roebuck, J. W., and Crowley, J. H.: A method of studying leukouytic functions in rive, Ann. N. Y. Acad. Sci., 59:757, 1955. 10. Roebuck, J. W,, Monte, R. W., Monagham, E. A., and Riddle, J. M.: Potentialities of the lymphocyte with an additional reference to its dysfunction in Hodgkin's disease. Ar~n. N, Y. Acad, Sci., 73:8, 1958. 11. Russel, R. ]., .Wilkinson, P. C., Sless, F., and Parrot, O. M.: Chemotaxfs of lymphob[ast. Nature (Lend}., 256:646, 1975. 12. Schmalzl, F., Huber, H., Asamer, K., Abbrederis, K., and Braunsteiner, H.: Cytochemical and imnmnohistologic investigations on the source and the functional changes of mononuclear cells in skin window exudates. Blood, 34:129, 1969. 13. Schreiner, G. F., and Unanue, E. R.: Anfi-Ig triggered movements of lymphocytes; specificity and lack of evidence for directional migration. J. Immunol., 114:809, 1975. 14. Smith, P. B., Tomfohrde, K. M., Rhoden, D. L., and Balows, A.: APt system, a multitube micromethod for identification of Enterobacteriaceae. Appl. Microbiol., 24:449-452, 1972. 15. Trepel, F., end Begemann, H.: On the origin of the skin window macrophages. Aeta Haomato[., 36:386, 1966. 16. Van Furth, R.'. Origin and kinetics of monocytes end macrophages. Semin. Hamnatol., 7:125, 1970. 17. Wilkinson, P. C., Roberts, J. A., Russel, R. J., and McLoughlin, M.: Chemotaxis of mitogen-activated human lymphocytes and the effect of membrane active enzymes. Clin. Exp. Immuno[., 25:289, 1976. 18. Zakrzewski, A.: On the importance of trace elements for mucosa of upper air passages. Acta Otolaryngol., 65:55-58, 1968.
Acknowledgment The authors are grateful to Mr. I. Boxall, Principal Scientific Laboratory Officer of the L o n d o n Institute of
HUSSEIN H. TOPPOZADA ET AL.
19 Midan Sahad Zaghloul Alexandria Egypt (Dr. Toppozada)
35