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The Immunological Features of Peyronie's Disease
0022-5347/96/155 1-0159$03.00/0 Tiik .JoL!KNAI. OF UROLOGY Copyright 0 1996 by AMERICAN URUI,OGICN. ASSWIATION, INC
Vol. 155, 159-162, January 1996 Printed in U S A .
THE IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE DAVID J. RALPH, RITA MIRAKIAN, JOHN P. PRYOR
ANL)
GIAN F. BOTTAZZO
F’IYJII~Sl. f‘(>lcrtvH(~spit01.Drparfriit,rit of Iinniunology. Royal L V I I ~ ( Hospital JII and Iiistitule of’
~ J r o / i ~ ,Limdoii. ~p,
Eirglar~tl
ABSTRACT
P u r p o s e : We investigated the immunological f e a t u r e s a n d possible a u t o i m m u n e b a s i s of Peyronie’s disease. Materials and Methods: The sera of 100 patients with Peyronie’s disease were tested for circulating autoantibodies, including anti-penis antibodies, by indirect immunofluorescence. Antibody deposition a n d the immunological activity in Peyronie’s plaque tissue from patients with early a n d long-standing disease were also assessed. Results: Circulating anti-penis antibodies were not found in a n y patient although antinuclear antibodies w e r e present i n 24%. Patients w i t h early Peyronie’s disease had IgM antibody deposition, marked T lymphocytic and macrocytic infiltration in the sub-tunical space, increased expression of adhesion molecules by endothelial cells a n d an increased human lymphocyte antigen class 2 expression by the cellular infiltrate, indicating cellular i m m u n e activation. Conclusions: These results show that some of the features of autoimmunity, in particular the cell mediated response, are present in Peyronie’s disease. KEYWORDS:penile induration, penis, penile erection, immunology
Peyronie’s disease is a disorder of unknown etiology characterized by the appearance of an inflammatory cell infiltrate in the sub-tunical space in the early active stage of the disease leading to fibrosis in the latter stage.1.2 The cause of the inflammation has not been established but it may include repeated minor sexual trauma3.4 or infection,5 or the condition may be part of a more generalized fibrosis as shown by the association with other fibrotic diseases, most notably Dupuytren’s contracture in 15% of the patients.6 If patients have a n inherited predisposition to the disease due to a particular tissue type,4 then an immunological component of the disease with a possible autoimmune basis may be cont r i b u t o r ~We . ~ addressed this suggestion by investigating the humoral and cellular immune systems in patients with Peyronie’s disease. PATIENTS AND METHODS
The sera of 100 patients with Peyronie’s disease were used for serological and hematological testing in varying numbers when appropriate. Mean patient age was 55 years (range 29 to 76). Other relevant diseases present included rheumatoid arthritis in 3 patients, gout in 8, diabetes in 11 and Dupuytren’s contracture in 27. No other autoimmune diseases were present in the patients tested. The ABO and rhesus (Rh) factor blood group antigens were assessed using a standard technique. Serological markers. The erythrocyte sedimentation rate measured by the Westergren method, C-reactive protein, serum immunoglobulins (IgA, IgG and IgM), and complement fractions C3 and C4 measured by rate nephelometry were all assessed as possible markers of immune activation. Antineutrophil cytoplasmic antibodies were also assessed by indirect immunofluorescence.8 These investigations were performed predominately on patients with either suspected or biopsy proved active disease. Autoantibody screen. Using indirect immunofluorescences the sera were screened for organ-specific autoantibodies USing human thyroid, pancreas and stomach. Nonorgan-specific antibodies were detected using rat liver and kidney as a substrate for the identification of human antinuclear, antismooth muscle and anti-mitochondria1 antibodies.
Rheumatoid factor and anti-cartilage antibodies. Rheumatoid factor was assessed using latex and then confirmed by the more accurate rheumatoid arthritis hemagglutination test. Since fibrosis of the cartilage of the external ear has been reported in Peyronie’s disease,lo anti-cartilage antibodies were investigated by indirect immunofluorescence using rat cartilage as a substrate and aided by hyaluronidase digestion to increase the available antigenic sites. Anti-penis antibodies. To test for autoantibodies directed against structures within the penis, indirect immunofluorescence was performed using 4 pm. cryostat sections of normal fresh penis obtained from a transsexual with blood group 0. The sections were incubated first with patient sera and then with a conjugate of sheep anti-human immunoglobulin. Humoral and cellular immune investigations. To investigate the cellular immune system in Peyronie’s disease and the possibility of antibody deposition, tissues from the plaque of 12 patients with acute Peyronie’s disease (less than 3 months in duration) and 5 with disease longer than 15 months in duration were collected. These specimens were compared to the “normal” tunica of 11 patients with Peyronie’s disease and 5 controls with a congenital deformity undergoing correction via the Nesbit technique. With the patient under local anesthesia small elliptical biopsies of the acute plaque were obtained. The tissue was snap frozen and the cryostat sections were prepared. Antibody deposition was detected by direct immunofluorescence using sheep anti-human IgA, IgG or IgM, or complement C3 conjugated with fluorescein isothiocyanate. The nature and immunological properties of the cellular infiltration were assessed by indirect immunofluorescence using monoclonal antibody to T lymphocytes and subsets (UCHT1, CD4 and CD8), macrophages (EB11) and B lymphocytes (LCD19 and BC3). Human leukocyte antigen class 2 expression (Mid 3), and the intracellular adhesion molecule (ICAM-l), vascular adhesion molecule (VCAM-l), endothelial leukocyte adhesion molecule (ELAM-1) and granulocyte membrane protein (GMP-140) were also investigated. A monoclonal antibody to factor 8 was used as an endothelial marker and universal endothelial antigen (UEA-1) labeled biotin with Texas red labeled avidin was used for double immunofluorescence. Mast cells were studied with toluidine blue.
Acce ted for publication April 13, 1995. Reat’ at annual meeting of American Urological Association, Washington, D. C., May 10-14, 1992. 159
160
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE TABLE2. Results of serological tests in Peyronie’s disease
RESULTS
Blood group antigens were assessed in 89 patients (table 1).Compared to 100 controls there was no significant difference between the ABO (p >0.1) or Rh (p >0.1) blood groups and the normal population. The d t a of the serologkd markers are shown in table 2. Many patients with an increased erythrocyte sedimentation rate or C-reactive protein were also known to suffer from arthritis, which may have accounted for the increased value. Some patients with biopsy proved active disease had normal parameters and so it was considered that erythrocyte sedimentation rate and C-reactive protein were not reliable markers of disease activity. The anti-neutrophil cytoplasmic antibodies, measured only in patients with known active disease, were negative in all cases. There was hypergammaglobulinemia in 13 patients, predominately IgG, and hypogammaglobulinemia in 4 with active disease. One or more circulating autoantibodies were present in 43 patients and 11 were positive for rheumatoid factor (table 3). Anti-cartilage antibodies were not detected in the 52 patients tested and no anti-penis antibodies were detected in any patient. The resulta using direct immunofluorescencerevealed the presence of IgM antibodies deposited within the sub-tunical layer of the acute plaques only. IgA, I g G and complement C3 antibodies were not found. A mononuclear cell infiltration was present within the acute plaque of 9 patients and within the plaque of 1 with long-standing disease. The infiltration was predominately within the sub-tunical space in close association with small blood vessels, and extending into the tunica itself in a perivascular fashion and into the erectile tissue (fig. 1). The infiltration consisted mainly of macrophagea and T lymphocytes with a T helper-to-T suppressor cell ratio of 2.3:l. The occaeional mast cell was detected within the infiltrate and few B lymphocytes were present. Angiogenesie was a feature in the inflamed plaques as demonstrated by increased endothelial factor 8 expression. AU acute plaques and the inflamed chronic plaque demonstrated a markedly increased human lymphocyte antigen class 2 expression by the inflammatory infiltrate and endothelium of the tunid and sub-tunical vessels (fig. 2,A and B). Antigen presenting cells (mainly dendritic cells) were present within the tunica and also expressed the human lymphocyte antigen class 2 antigen (fig. 2, C).The control tissue, Nesbit ellipses and 4 chronic plaques did not contain anidlammatory infiltrate and had minimal human lymphocyte antigen class 2 expression. Adhesion molecule expression is summarized in table 4. There was marked expression of these molecules, in particular GMP-140and ICAM-1,by the endothelium of large vessels in the tunica, sub-tunical space and subcutaneous tissue of acute Peyronie’s plaques (fig. 2,D). DISCUSSION
The role of the immune system in Peyronie’s disease has been evaluated for possible markers of disease activity and autoantibodies to assess a possible autoimmune basis for the disease. The nature,site and immunological properties of the
No. Parameter
No. Elevated
NO. Decreased
18
5
-
65
9 3 8 2
-
pts.
Tested
Erythrocyte sedimentation rate Creadive pmtaii
68 68
IgA
IgG IgM Complement C3 Complement C4 Anti-neutmphil cytoplasmic an-
68
2 1
1
-
2 2 AU neg.
56 56
18
1
tihndies
TABLE3. Circulating autoantibodies in 100 patients with Peyronie’s disease compared to age and sex-matched controls No. Pta.
Controls
Organ-specit2 antibodies Parietal cell Thyroglobulin Thyroid microwmal Islet cell
2 6 6 3
4
2 10 0
Nonorgan-specificantibodies 24 Antinuclear antibodies 3 Mitnchondrial 7 Smooth muscle Rheumatoid factor 11 * p <0.001.
4% Less than 1 3 5
cellular infiltrate throughout the course of the disease were also noted. The management of Peyronie’s disease would be greatly improved if the level of disease activity was known. Such knowledge would allow medical treatment to be instigated only in the active stage of disease and surgical correction of the penile deformity in the inactive stage, with improved results. The erythrocyte sedimentation rate and C-reactive protein were elevated in a few patients with active disease who also had other conditions that could have accounted for the elevation. However, some patients with biopsy proved active disease had normal parameters and so it was considered that erythrocyte sedimentation rate and C-reactive protein were unreliable markers of disease activity. There was hypergammaglobulinemia in 13 patients and hypogammaglobulinemia in 4. This condition occurred only in patients with active disease, which may be a reflection of the systemic reaction to the localized penile inflammation and, therefore, a more reliable measure of disease activity. It has been suggested that there is vasculitis of the small sub-tunical vessels in Peyronie’s disease.’ Therefore, antineutrophil cytoplasmic antibodies, proved markers of vasculitis in other diseases, were assessed in patients with known active disease.” The test for these antibodies was negative, which suggests that the vasculitis was either a type negative for anti-neutrophil cytoplasmic antibodies or that these antibodies are not a feature of Peyronie’s disease. Because autoimmune diseases tend to overlap, the presence of autoantibodies specific to other organs or to nonspe-
TAEIL~?~ 1. Blood group antigens in 89 patients with Peyronie’s TABU4. Adhesion molecule expression in Peyronie’s disease~-
disease and 100 controls
0 A B
AB Rh pa. nso.
62 (Ml.4) 29 (32.6) 7 (7.9)
47 42
Adhesion
Molecule
Plaque
8
1 (1.1)
3
79 (88.8) lO(11.2)
85 15
Graded (1+-6+) Expression of Tissue Groups
Acute
GMP-140 ICAM-1 VCAM-1 ELAM-1
++++++ ++++ ++
++
Chronic Plaque
Nesbit Ellipse
++ + + +
+
+ + -
+ + +
-
-
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE
161
~-
FIG. 1. Indirect immunofluorescent stains of acute Peyronie’splaque.A, T lymphocytes within sub-tunid space. Reduced from ~ 4 0 0B, . double immunofluorescence shows macrophage (green) iniiltration around small blood vessels (red) in sub-tunical space (St). T,tunica. E, erectile tissue. Reduced from X250. C, double immunofluorescence of macrophages within sub-tunid space extending along cavernous septa (Cs).Reduced from X250. D, double hmunofluorescenceofedge of plaque with minimalinfiltrationwithin sub-tunid layer. R e d u c e d from X250.
cific cellular components may suggest an autoimmune basis viously to good effect by treating the inflammatory stage of in Peyronie’s disease. Of the 100 patients tested 43 had 1or Peymnie’s disease with tamoxifen, a drug known to inhibit more circulating autoantibodies. Of sigmficance was that the action of transforming growth factor 0 in vitro.12 The 24% of the patients were positive for antinuclear antibody (p fibrogenesis, which will persist as long as the antigen is still <0.001) and to a lesser extent 11%were positive for rheu- present, occurs at the site of the inflammatory infiltration. matoid factor, which is a higher rate than expected for mid- Since this infiltration has been shown to extend into the dle-aged men. Because these features are common in other erectile tissue (fig. 2, A), a degree of cavernous fibrosis will autoimmune connective tissue diseases, this Snding would occur that may, if severe, lead to distal penile flaccidity.13 Inappropriate epithelial human lymphocyte antigen class 2 suggest that Peyronie’s disease might be similar. Although antibodies directed at the constituents of the expression has been shown to be a feature of autoimmune penis were not circulating in the patient serum, IgM anti- disease.14 which may occur in Peyronie’s disease with the bodies were deposited within the sub-tunical layer of the endothelial class 2 expression. In the area of inflammation, endothelial cells express a plaque in patients with active disease. The use of more sensitive enzyme-linked assays using extracts of penis may in range of molecules known as cellular adhesion molecules.16 the future detect circulating anti-penis antibodies, which Circulating leukocytes display receptors for these molecules then would correlate with #is finding of antibody deposition that, therefore, allow them to home in at the site of inflammation. By studying the adhesion molecule expression in within the plaque. The cellular infiltration of the acute plaques consisted Peyroniek disease, the site of maximal disease activity could mainly of T lymphocytes and macrophages, predominately in be seen. All adhesion molecules tested were expressed by the the sub-tunid space surrounding small blood vessels but endothelium in active Peyronie’s disease, in particular GMPalso within the tunica albuginea in a perivascdar fashion 140. The expression was noted on vessels within the sub(fig. 1, A and B). The infiltration also extended into the tunical space and tunica, and in some patients the superficial erectile tissue and along the cavernous septa (fig. 1, The tissue (fig. 2, D).Therefore, these experiments would suggest infiltration, endothelium of the tunica and sub-tunical ves- that the inflammation is transmural in nature. Many autosels, and antigen-presenting dendritic cells within the tunica immune diseeses are associated with specific tissue types, expressed the human lymphocyte antigen class 2 antigen which is suggestive of a genetic predisposition. Therefore, the (fig.2, A to C).The T helper lymphocytesare able to recognize association of the human lymphocyte antigen B7 cross-reactantigen on antigen-presenting cells when presented in asso- ing antigens in Peyronie’s disease’s adds support to an imciation with class 2 molecules, which then leads to activation munological component. Blood group antigens may also show of cytokines released from the T lymphocytes and macro- a genetic predisposition to disease susceptibility and, therephages with the promotion of fibrogenesis. Inhibition of ~ y - fore, they were assessed in Peyronie’s disease. Although 58% no statistically significant tokine action, in particular transforming growth factor B, ofthe patients had blood group 0, could therefore dewease the fibrogenesis and prevent pro- association was detected between blood group antigens and gression of the disease. This hypothesis has been tested p ~ -Peymnie’s disease.
c).
162
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE
FIG.2. Indirect immunofluorescent stains of acute Peymnie’s p%ue. A, human lymph-
antigen class 2 e ression by cellular
infiltrateand endothelium (arrow) within sub-tunicalspace (St)extendmgintotunica (T) and erectde.hssue (E).Reduc3from X250. B , high
(r)
cation of human lymphocyte antigen class 2 expression of cellular infiltrate and endothehum (arrow) m t h m sub-tunical s ace within tunica albuginea. Refuced from X400. C, human 1 phocyte antigen class 2 expression of antigen presenting cells €ram X250. D, granulocyte memr-e protein expression of subtunid and tunical endothelium. duced from X250.
CONCLUSIONS
Our study revealed many features that add support to an autoimmune basis for Peymnie’s disease, includingthe presence of circulating antinuclear antibodies, hypergammaglobulinemia, antibody deposition within the plaque and cellmediated immunity as shown by an increased human lymphocyte antigen class 2 expression within the plaque. However, the antigen that triggers the inflammatory response and the presence of circulating anti-penis antibodies have not been detected with the techniques used. Therefore, the u8e of more sensitive assays concentrating on the cellmediated response will be needed in the future to determiue if an autoirnmune component is present. “he nature of the inflammation,however, being cell-mediated with T lymphocytes and mamphages, usually occurs in response to an antigen and is not what is expected in primary wound healing due to trauma. If trauma does have a role in Peymnie’s disease,then it is likely that it c a w s exposure of the antigen that eta up the inflammatory response, which would suggest that the antigen is a constituent of the penis and therefore, it should, make future detection easier. REFERENCES
1. Smith, B. H.: Peyronie’s disease.h e r . J. Clin. Path., 45:670,1966. 2. Bystrlim, J. and Rubio, C.: Induratio penis plastica Peyronie’s
Int., 35.407, 1980. 5. de la Peyronie, F.: Sur quelques obstacles qui s’opposent a l’ejaculation naturelle de la semence. Mem. Acad. Chir., p. 425, 1743. 6. Chilton, C. P., Castle, W. M.,Westwood, C. A. and Pryor, J. P.: Factors associated in the aetiology of Peyronie’s disease. Brit. J. Urol.,84: 748,1982. 7. Chesney, J.: Peyronie’s disease. Brit. J. Urol., 47: 209, 1975. 8. Cambridge, G., Rampton, D. S., Stevens, T. R., McCarthy, D. A,, Kamm, M. and Leaker, J.: Anti-neutmphil antibodies in inflammatory bowel disease: prevalence and diagnostic role. Gut, 3 3 668, 1992. 9. Coons, A. M.: Fluorescent antibody methods. General Cytochem. Methods, 1:399, 1958. 10. Gallizia, M. F.: A collagen triad La Peyronie’s dmease, Dupuytren’s disease and fibrosis of the auricular cartilage. J. d’ Urol. Nephrol., 7 0 424, 1964. 11. Jennette, J. C. and Falk, R. J.: Diagnostic classification of antineutmphil cytoplasmic autoantibody-associatedvasculitides. 184, 1991. h e r . J. Kidney Dis., 1%: 12. Ralph, D. J., Brooks,M. D., Bottazzo. G. F. and Prvor. J. P.: The treatment of Peymnie’s disease with tamoxifen.-Brit. J. Urol., 70: 648,1992. 13. Ralph, D. J.,Hughes, T., Lees, W. R. and Pryor, J. P.: Pre-operative assessment of Peyronie’s disease using colour Doppler sonography. Brit. J. Urol., 6 9 629, 1992. 14. Bottazzo, G. F., Pujol-Borrell, R., Hanafusa, T. and Feldmann,
M.: Role of aberrant HLA-DR expression and antigen presendisease. Clinical featurea and etiology. Scand. J. Urol. Nephtation in induction of endocrine autoimmunity. Lancet, 2 rol., 10: 12, 1976. 1115,1983. 3. Vande Berg, J. S., Devine, C. J.,Jr., Horton, C. E., Somers, K. D., 15. Osborn, L.: Leukocyte adhesion to endothelium in inflammation. Wright, G. L., Jr., Leffell, M. S., Dawson, D. M.,Gleischman, Cell, 6 2 3, 1990. S.H.and Rowe, M.J.: Mechanisms of calci6cationin Peyronie’s 16. Willscher. M. K. Cwazka. W. F. and Novicki. D. E.: The associdisease. J. Urol., la?:52, 1982. ation of histkompatibiiity antigens of the’ B7 cross-reacting 4. Hinman. F.. Jr.: Etiolonic factors in Pevronie’s rl. .~ ~ disease. . _ U _._. group with Peyronie’s disease. J. Urol., 122 34, 1979. I
.
0
Vol. 155, 159-162, January 1996 Printed in U S A .
THE IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE DAVID J. RALPH, RITA MIRAKIAN, JOHN P. PRYOR
ANL)
GIAN F. BOTTAZZO
F’IYJII~Sl. f‘(>lcrtvH(~spit01.Drparfriit,rit of Iinniunology. Royal L V I I ~ ( Hospital JII and Iiistitule of’
~ J r o / i ~ ,Limdoii. ~p,
Eirglar~tl
ABSTRACT
P u r p o s e : We investigated the immunological f e a t u r e s a n d possible a u t o i m m u n e b a s i s of Peyronie’s disease. Materials and Methods: The sera of 100 patients with Peyronie’s disease were tested for circulating autoantibodies, including anti-penis antibodies, by indirect immunofluorescence. Antibody deposition a n d the immunological activity in Peyronie’s plaque tissue from patients with early a n d long-standing disease were also assessed. Results: Circulating anti-penis antibodies were not found in a n y patient although antinuclear antibodies w e r e present i n 24%. Patients w i t h early Peyronie’s disease had IgM antibody deposition, marked T lymphocytic and macrocytic infiltration in the sub-tunical space, increased expression of adhesion molecules by endothelial cells a n d an increased human lymphocyte antigen class 2 expression by the cellular infiltrate, indicating cellular i m m u n e activation. Conclusions: These results show that some of the features of autoimmunity, in particular the cell mediated response, are present in Peyronie’s disease. KEYWORDS:penile induration, penis, penile erection, immunology
Peyronie’s disease is a disorder of unknown etiology characterized by the appearance of an inflammatory cell infiltrate in the sub-tunical space in the early active stage of the disease leading to fibrosis in the latter stage.1.2 The cause of the inflammation has not been established but it may include repeated minor sexual trauma3.4 or infection,5 or the condition may be part of a more generalized fibrosis as shown by the association with other fibrotic diseases, most notably Dupuytren’s contracture in 15% of the patients.6 If patients have a n inherited predisposition to the disease due to a particular tissue type,4 then an immunological component of the disease with a possible autoimmune basis may be cont r i b u t o r ~We . ~ addressed this suggestion by investigating the humoral and cellular immune systems in patients with Peyronie’s disease. PATIENTS AND METHODS
The sera of 100 patients with Peyronie’s disease were used for serological and hematological testing in varying numbers when appropriate. Mean patient age was 55 years (range 29 to 76). Other relevant diseases present included rheumatoid arthritis in 3 patients, gout in 8, diabetes in 11 and Dupuytren’s contracture in 27. No other autoimmune diseases were present in the patients tested. The ABO and rhesus (Rh) factor blood group antigens were assessed using a standard technique. Serological markers. The erythrocyte sedimentation rate measured by the Westergren method, C-reactive protein, serum immunoglobulins (IgA, IgG and IgM), and complement fractions C3 and C4 measured by rate nephelometry were all assessed as possible markers of immune activation. Antineutrophil cytoplasmic antibodies were also assessed by indirect immunofluorescence.8 These investigations were performed predominately on patients with either suspected or biopsy proved active disease. Autoantibody screen. Using indirect immunofluorescences the sera were screened for organ-specific autoantibodies USing human thyroid, pancreas and stomach. Nonorgan-specific antibodies were detected using rat liver and kidney as a substrate for the identification of human antinuclear, antismooth muscle and anti-mitochondria1 antibodies.
Rheumatoid factor and anti-cartilage antibodies. Rheumatoid factor was assessed using latex and then confirmed by the more accurate rheumatoid arthritis hemagglutination test. Since fibrosis of the cartilage of the external ear has been reported in Peyronie’s disease,lo anti-cartilage antibodies were investigated by indirect immunofluorescence using rat cartilage as a substrate and aided by hyaluronidase digestion to increase the available antigenic sites. Anti-penis antibodies. To test for autoantibodies directed against structures within the penis, indirect immunofluorescence was performed using 4 pm. cryostat sections of normal fresh penis obtained from a transsexual with blood group 0. The sections were incubated first with patient sera and then with a conjugate of sheep anti-human immunoglobulin. Humoral and cellular immune investigations. To investigate the cellular immune system in Peyronie’s disease and the possibility of antibody deposition, tissues from the plaque of 12 patients with acute Peyronie’s disease (less than 3 months in duration) and 5 with disease longer than 15 months in duration were collected. These specimens were compared to the “normal” tunica of 11 patients with Peyronie’s disease and 5 controls with a congenital deformity undergoing correction via the Nesbit technique. With the patient under local anesthesia small elliptical biopsies of the acute plaque were obtained. The tissue was snap frozen and the cryostat sections were prepared. Antibody deposition was detected by direct immunofluorescence using sheep anti-human IgA, IgG or IgM, or complement C3 conjugated with fluorescein isothiocyanate. The nature and immunological properties of the cellular infiltration were assessed by indirect immunofluorescence using monoclonal antibody to T lymphocytes and subsets (UCHT1, CD4 and CD8), macrophages (EB11) and B lymphocytes (LCD19 and BC3). Human leukocyte antigen class 2 expression (Mid 3), and the intracellular adhesion molecule (ICAM-l), vascular adhesion molecule (VCAM-l), endothelial leukocyte adhesion molecule (ELAM-1) and granulocyte membrane protein (GMP-140) were also investigated. A monoclonal antibody to factor 8 was used as an endothelial marker and universal endothelial antigen (UEA-1) labeled biotin with Texas red labeled avidin was used for double immunofluorescence. Mast cells were studied with toluidine blue.
Acce ted for publication April 13, 1995. Reat’ at annual meeting of American Urological Association, Washington, D. C., May 10-14, 1992. 159
160
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE TABLE2. Results of serological tests in Peyronie’s disease
RESULTS
Blood group antigens were assessed in 89 patients (table 1).Compared to 100 controls there was no significant difference between the ABO (p >0.1) or Rh (p >0.1) blood groups and the normal population. The d t a of the serologkd markers are shown in table 2. Many patients with an increased erythrocyte sedimentation rate or C-reactive protein were also known to suffer from arthritis, which may have accounted for the increased value. Some patients with biopsy proved active disease had normal parameters and so it was considered that erythrocyte sedimentation rate and C-reactive protein were not reliable markers of disease activity. The anti-neutrophil cytoplasmic antibodies, measured only in patients with known active disease, were negative in all cases. There was hypergammaglobulinemia in 13 patients, predominately IgG, and hypogammaglobulinemia in 4 with active disease. One or more circulating autoantibodies were present in 43 patients and 11 were positive for rheumatoid factor (table 3). Anti-cartilage antibodies were not detected in the 52 patients tested and no anti-penis antibodies were detected in any patient. The resulta using direct immunofluorescencerevealed the presence of IgM antibodies deposited within the sub-tunical layer of the acute plaques only. IgA, I g G and complement C3 antibodies were not found. A mononuclear cell infiltration was present within the acute plaque of 9 patients and within the plaque of 1 with long-standing disease. The infiltration was predominately within the sub-tunical space in close association with small blood vessels, and extending into the tunica itself in a perivascular fashion and into the erectile tissue (fig. 1). The infiltration consisted mainly of macrophagea and T lymphocytes with a T helper-to-T suppressor cell ratio of 2.3:l. The occaeional mast cell was detected within the infiltrate and few B lymphocytes were present. Angiogenesie was a feature in the inflamed plaques as demonstrated by increased endothelial factor 8 expression. AU acute plaques and the inflamed chronic plaque demonstrated a markedly increased human lymphocyte antigen class 2 expression by the inflammatory infiltrate and endothelium of the tunid and sub-tunical vessels (fig. 2,A and B). Antigen presenting cells (mainly dendritic cells) were present within the tunica and also expressed the human lymphocyte antigen class 2 antigen (fig. 2, C).The control tissue, Nesbit ellipses and 4 chronic plaques did not contain anidlammatory infiltrate and had minimal human lymphocyte antigen class 2 expression. Adhesion molecule expression is summarized in table 4. There was marked expression of these molecules, in particular GMP-140and ICAM-1,by the endothelium of large vessels in the tunica, sub-tunical space and subcutaneous tissue of acute Peyronie’s plaques (fig. 2,D). DISCUSSION
The role of the immune system in Peyronie’s disease has been evaluated for possible markers of disease activity and autoantibodies to assess a possible autoimmune basis for the disease. The nature,site and immunological properties of the
No. Parameter
No. Elevated
NO. Decreased
18
5
-
65
9 3 8 2
-
pts.
Tested
Erythrocyte sedimentation rate Creadive pmtaii
68 68
IgA
IgG IgM Complement C3 Complement C4 Anti-neutmphil cytoplasmic an-
68
2 1
1
-
2 2 AU neg.
56 56
18
1
tihndies
TABLE3. Circulating autoantibodies in 100 patients with Peyronie’s disease compared to age and sex-matched controls No. Pta.
Controls
Organ-specit2 antibodies Parietal cell Thyroglobulin Thyroid microwmal Islet cell
2 6 6 3
4
2 10 0
Nonorgan-specificantibodies 24 Antinuclear antibodies 3 Mitnchondrial 7 Smooth muscle Rheumatoid factor 11 * p <0.001.
4% Less than 1 3 5
cellular infiltrate throughout the course of the disease were also noted. The management of Peyronie’s disease would be greatly improved if the level of disease activity was known. Such knowledge would allow medical treatment to be instigated only in the active stage of disease and surgical correction of the penile deformity in the inactive stage, with improved results. The erythrocyte sedimentation rate and C-reactive protein were elevated in a few patients with active disease who also had other conditions that could have accounted for the elevation. However, some patients with biopsy proved active disease had normal parameters and so it was considered that erythrocyte sedimentation rate and C-reactive protein were unreliable markers of disease activity. There was hypergammaglobulinemia in 13 patients and hypogammaglobulinemia in 4. This condition occurred only in patients with active disease, which may be a reflection of the systemic reaction to the localized penile inflammation and, therefore, a more reliable measure of disease activity. It has been suggested that there is vasculitis of the small sub-tunical vessels in Peyronie’s disease.’ Therefore, antineutrophil cytoplasmic antibodies, proved markers of vasculitis in other diseases, were assessed in patients with known active disease.” The test for these antibodies was negative, which suggests that the vasculitis was either a type negative for anti-neutrophil cytoplasmic antibodies or that these antibodies are not a feature of Peyronie’s disease. Because autoimmune diseases tend to overlap, the presence of autoantibodies specific to other organs or to nonspe-
TAEIL~?~ 1. Blood group antigens in 89 patients with Peyronie’s TABU4. Adhesion molecule expression in Peyronie’s disease~-
disease and 100 controls
0 A B
AB Rh pa. nso.
62 (Ml.4) 29 (32.6) 7 (7.9)
47 42
Adhesion
Molecule
Plaque
8
1 (1.1)
3
79 (88.8) lO(11.2)
85 15
Graded (1+-6+) Expression of Tissue Groups
Acute
GMP-140 ICAM-1 VCAM-1 ELAM-1
++++++ ++++ ++
++
Chronic Plaque
Nesbit Ellipse
++ + + +
+
+ + -
+ + +
-
-
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE
161
~-
FIG. 1. Indirect immunofluorescent stains of acute Peyronie’splaque.A, T lymphocytes within sub-tunid space. Reduced from ~ 4 0 0B, . double immunofluorescence shows macrophage (green) iniiltration around small blood vessels (red) in sub-tunical space (St). T,tunica. E, erectile tissue. Reduced from X250. C, double immunofluorescence of macrophages within sub-tunid space extending along cavernous septa (Cs).Reduced from X250. D, double hmunofluorescenceofedge of plaque with minimalinfiltrationwithin sub-tunid layer. R e d u c e d from X250.
cific cellular components may suggest an autoimmune basis viously to good effect by treating the inflammatory stage of in Peyronie’s disease. Of the 100 patients tested 43 had 1or Peymnie’s disease with tamoxifen, a drug known to inhibit more circulating autoantibodies. Of sigmficance was that the action of transforming growth factor 0 in vitro.12 The 24% of the patients were positive for antinuclear antibody (p fibrogenesis, which will persist as long as the antigen is still <0.001) and to a lesser extent 11%were positive for rheu- present, occurs at the site of the inflammatory infiltration. matoid factor, which is a higher rate than expected for mid- Since this infiltration has been shown to extend into the dle-aged men. Because these features are common in other erectile tissue (fig. 2, A), a degree of cavernous fibrosis will autoimmune connective tissue diseases, this Snding would occur that may, if severe, lead to distal penile flaccidity.13 Inappropriate epithelial human lymphocyte antigen class 2 suggest that Peyronie’s disease might be similar. Although antibodies directed at the constituents of the expression has been shown to be a feature of autoimmune penis were not circulating in the patient serum, IgM anti- disease.14 which may occur in Peyronie’s disease with the bodies were deposited within the sub-tunical layer of the endothelial class 2 expression. In the area of inflammation, endothelial cells express a plaque in patients with active disease. The use of more sensitive enzyme-linked assays using extracts of penis may in range of molecules known as cellular adhesion molecules.16 the future detect circulating anti-penis antibodies, which Circulating leukocytes display receptors for these molecules then would correlate with #is finding of antibody deposition that, therefore, allow them to home in at the site of inflammation. By studying the adhesion molecule expression in within the plaque. The cellular infiltration of the acute plaques consisted Peyroniek disease, the site of maximal disease activity could mainly of T lymphocytes and macrophages, predominately in be seen. All adhesion molecules tested were expressed by the the sub-tunid space surrounding small blood vessels but endothelium in active Peyronie’s disease, in particular GMPalso within the tunica albuginea in a perivascdar fashion 140. The expression was noted on vessels within the sub(fig. 1, A and B). The infiltration also extended into the tunical space and tunica, and in some patients the superficial erectile tissue and along the cavernous septa (fig. 1, The tissue (fig. 2, D).Therefore, these experiments would suggest infiltration, endothelium of the tunica and sub-tunical ves- that the inflammation is transmural in nature. Many autosels, and antigen-presenting dendritic cells within the tunica immune diseeses are associated with specific tissue types, expressed the human lymphocyte antigen class 2 antigen which is suggestive of a genetic predisposition. Therefore, the (fig.2, A to C).The T helper lymphocytesare able to recognize association of the human lymphocyte antigen B7 cross-reactantigen on antigen-presenting cells when presented in asso- ing antigens in Peyronie’s disease’s adds support to an imciation with class 2 molecules, which then leads to activation munological component. Blood group antigens may also show of cytokines released from the T lymphocytes and macro- a genetic predisposition to disease susceptibility and, therephages with the promotion of fibrogenesis. Inhibition of ~ y - fore, they were assessed in Peyronie’s disease. Although 58% no statistically significant tokine action, in particular transforming growth factor B, ofthe patients had blood group 0, could therefore dewease the fibrogenesis and prevent pro- association was detected between blood group antigens and gression of the disease. This hypothesis has been tested p ~ -Peymnie’s disease.
c).
162
IMMUNOLOGICAL FEATURES OF PEYRONIE’S DISEASE
FIG.2. Indirect immunofluorescent stains of acute Peymnie’s p%ue. A, human lymph-
antigen class 2 e ression by cellular
infiltrateand endothelium (arrow) within sub-tunicalspace (St)extendmgintotunica (T) and erectde.hssue (E).Reduc3from X250. B , high
(r)
cation of human lymphocyte antigen class 2 expression of cellular infiltrate and endothehum (arrow) m t h m sub-tunical s ace within tunica albuginea. Refuced from X400. C, human 1 phocyte antigen class 2 expression of antigen presenting cells €ram X250. D, granulocyte memr-e protein expression of subtunid and tunical endothelium. duced from X250.
CONCLUSIONS
Our study revealed many features that add support to an autoimmune basis for Peymnie’s disease, includingthe presence of circulating antinuclear antibodies, hypergammaglobulinemia, antibody deposition within the plaque and cellmediated immunity as shown by an increased human lymphocyte antigen class 2 expression within the plaque. However, the antigen that triggers the inflammatory response and the presence of circulating anti-penis antibodies have not been detected with the techniques used. Therefore, the u8e of more sensitive assays concentrating on the cellmediated response will be needed in the future to determiue if an autoirnmune component is present. “he nature of the inflammation,however, being cell-mediated with T lymphocytes and mamphages, usually occurs in response to an antigen and is not what is expected in primary wound healing due to trauma. If trauma does have a role in Peymnie’s disease,then it is likely that it c a w s exposure of the antigen that eta up the inflammatory response, which would suggest that the antigen is a constituent of the penis and therefore, it should, make future detection easier. REFERENCES
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Int., 35.407, 1980. 5. de la Peyronie, F.: Sur quelques obstacles qui s’opposent a l’ejaculation naturelle de la semence. Mem. Acad. Chir., p. 425, 1743. 6. Chilton, C. P., Castle, W. M.,Westwood, C. A. and Pryor, J. P.: Factors associated in the aetiology of Peyronie’s disease. Brit. J. Urol.,84: 748,1982. 7. Chesney, J.: Peyronie’s disease. Brit. J. Urol., 47: 209, 1975. 8. Cambridge, G., Rampton, D. S., Stevens, T. R., McCarthy, D. A,, Kamm, M. and Leaker, J.: Anti-neutmphil antibodies in inflammatory bowel disease: prevalence and diagnostic role. Gut, 3 3 668, 1992. 9. Coons, A. M.: Fluorescent antibody methods. General Cytochem. Methods, 1:399, 1958. 10. Gallizia, M. F.: A collagen triad La Peyronie’s dmease, Dupuytren’s disease and fibrosis of the auricular cartilage. J. d’ Urol. Nephrol., 7 0 424, 1964. 11. Jennette, J. C. and Falk, R. J.: Diagnostic classification of antineutmphil cytoplasmic autoantibody-associatedvasculitides. 184, 1991. h e r . J. Kidney Dis., 1%: 12. Ralph, D. J., Brooks,M. D., Bottazzo. G. F. and Prvor. J. P.: The treatment of Peymnie’s disease with tamoxifen.-Brit. J. Urol., 70: 648,1992. 13. Ralph, D. J.,Hughes, T., Lees, W. R. and Pryor, J. P.: Pre-operative assessment of Peyronie’s disease using colour Doppler sonography. Brit. J. Urol., 6 9 629, 1992. 14. Bottazzo, G. F., Pujol-Borrell, R., Hanafusa, T. and Feldmann,
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