- Email: [email protected]
Histologic, immunofluorescent, and antinuclear antibody findings in PUVA-treated patients
I
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Histologic, immunofluorescent, and antinuclear antibody findings in PUVA-treated patients Donald L. Levin, M.D., Henry H. Roenigk, Jr., M . D . , William A. Caro, M . D . , and Mary Lyons, M.D. Chicago, IL Twenty-two patients with psoriasis were treated with PUVA therapy for over two years. Skin biopsies from sun-exposed skin were evaluated before, during, and after PUVA therapy. No statistically significant epidermal changes were found when looking for hyperkeratosis, atrophy, melanin, or melanocyte nmnbers. No dyskeratosis was found. Dermal changes, including alterations in elastic tissue and collagen, were not statistically significant. Amyloid deposits were absent. The amount of mucin deposition was found to correlate with the length of PUVA therapy. In four patients, the mucin disappeared after withdrawal of PUVA, suggesting that this change is reversible. (J AM ACAD DERMATOL6:328-333, 1982.)
Photochemotherapy (PUVA), utilizing 8-methoxypsoralen (P) and long-wavelength ultraviolet light (UVA), has been used in the treatment of psoriasis since 1974.' It is an effective form of therapy" that is readily accepted by patients. The side effects of PUVA are well known 3 and include erythema, pruritus, nausea, formation of cutaneous tumors, and the possible development of cataracts. This prospective study will review the histopathologic findings in PUVA-treated patients and will present data on the direct immunofluorescent and antinuclear antibody (ANA) values in a series of patients with psoriasis treated with PUVA therapy. METHODS Twenty-two patients with psoriasis were randomly selected from the PUVA Center at Northwestern Uni-
From the Department of Dermatology, Northwestern University Medical School. Reprint requests to: Dr. Henry H. Roenigk, Jr., Northwestern University Medical School, Department of Dermatology, 303 East Chicago Ave., Chicago, IL 60611.
328
versity. All patients had psoriasis involving greater than 30% of the body surface area. All patients had been treated previously with topical steroids and ultraviolet B (UVB) therapy. Except for the four patients who discontinued treatment because of remission of their psoriasis, all patients continued to use topical corticosteroids in order to minimize their exposure to PUVA. The frequency of maintenance therapy was determined according to disease activity, in order to maintain clinical remission. Most patients on maintenance received treatments two to four times per month. 8-Methoxypsoralen (8-MOP) was ingested in doses of 0.5 to 0.6 mg/kg, according to weight and skin type, 2 hours before PUVA therapy. Patients were started at 1.0 joule/cm2; the dosage was increased until clearing of psoriasis was evident, according to standard methods. 2'3 Plateau method of giving PUVA 2 was used to minimize exposure to UVA. Four-millimeter skin biopsies for light microscopy and immunofluorescence were obtained from the upper arm (noninvolved, sun-exposed). Sections were stained with hematoxylin and eosin, iron, Congo red, and acid orcein-Giemsa according to standard procedures. Sections were examined without clinical information and without known order of sequence.
0190-9622/82/030328+06500.60/0 © 1982 Am Acad Dermatol
Volume 6 Number 3 March, 1982
hnmttnofluorescent and ANA findings in PUVA
329
T a b l e I. Patient population s u m m a r y , time of biopsy, age, and joules/cm 2 Biopsies (months after starting PUVA) Group
3 months
17-24 months
Age ± S.D.
Total joules/ cm z ± S.D,
x
x
x
x
x x
x x
x
x
47.6 ___ 14.8 50.1 ~ 13.9 35 59
2,219 _ 968 2,085 + 925 595 624
Number of patients
Pre-
I
10
II IIIA
8 2
IIIB
2
T a b l e II. E p i d e r m a l changes before, during, and after P U V A therapy
Hyperkeratosis Present Absent Atrophy Present Absent Melanocytes Decreased Normal Increased Melanin Decreased Normal Increased
Early
PrePUVA
PUVA
Late PUVA
PostPUVA
11 1
19 3
18 0
3 1
8 4
9 13
14 4
2 2
1 4 7
2 9 11
0 7 11
0 0 4
1 5 6
1 12 9
0 8 10
1 0 3
Biopsies for immunofluorescence were snap-frozen in liquid nitrogen, and specimens were stained with fluorescein-conjugated IgG, IgM, C3, and Clq, according to standard procedures. Serum ANA test was performed at the time of each skin biopsy. PATIENTS The psoriasis patients were divided into three groups (Table I). Groups I and II received continuous PUVA therapy, whereas group III went into remission with initial therapy; therefore, no further PUVA was given. Group I: Biopsy examination was done on ten patients (mean age, 47.6 ___ 14.8 years) before PUVA was initiated, 3 months later, and 2 years after PUVA was begun. Mean total UVA dosage was 2,219 ___968 joules/cm ~(mean +__S.D.). Group H: Biopsy examination was done on eight patients (mean age, 50.1 ___ 13.9 years) 3 months after PUVA therapy was initiated, and 2 years after PUVA therapy was begun. These patients did
not receive a pretreatment biopsy. Mean total UVA dosage was 2,085 + 925 joules/era 2 (mean ___ S.D.). Group IliA: Biopsies were done on two patients (average age, 35) prior to initiation of PUVA therapy, and 3 months after therapy was initiated. Due to remission at 6 months, PUVA therapy was terminated. Neither patient received PUVA therapy in the ensuing 18 months. Repeat skin biopsy was performed at 18 months. The average total dosage was 595 joules/era ~. Group IIIB: Biopsies were done on two additional patients (average age, 59) 3 months after initiation of therapy. These patients were treated until remission 4 months later and then were withdrawn from therapy. Biopsy examination was repeated 17 months after PUVA therapy ceased. The average total dosage was 624 joules/cm ~-.
330
Journal of the American Academy of Dermatology
L e v i n et al
Fig. 1. Epidermal changes showing hyperkeratosis, atrophy, increased melanocytes, and melanin content present in most biopsies. (Hematoxylin-eosin stain; x 400.)
Table III. Elastic tissue changes I -
Normal Decreased Fragmented Total
PrePUVA
Early PUVA
PUVA
PostPUVA
9 1 2 12
17
15
3
5 22
3 18
1 4
HISTOPATHOLOGY RESULTS
Epidermal changes Hyperkeratosis (Table II). Hyperkeratosis was present in the vast majority of biopsies examined at all stages of P U V A therapy. These changes were not statistically significant (p > 0.3) in any group (Fig. 1). A t r o p h y (Table II). Atrophy was present before PUVA, and late PUVA biopsies showed some increase in the number of patients with atrophy. In comparing all groups, there were no
Late
statistically significant differences (p > 0.1) between groups (Fig. 1). Dyskeratosis. We observed no evidence of dyskeratosis in any section studied at any time before, during, or after PUVA therapy (Fig. 1).
Basal layer melanocytes and melanin content (Table II). There were fewer melanocytes and less melanin in pretreatment biopsies than in early and late PUVA biopsies. There was a trend toward increased melanocytes and melanin content in early and late PUVA, but, comparing all groups
Volume 6 Number 3 March, 1982
hnrnunofluorescent and ANA findings in PUVA
MUCIN
MUCIN
LARGE
LARGE
MEDIUM
X
XX
Xx
SMALL
XX
xx
xx
•
xxx
NONE
•
•
x
•
xx
oe
xx
gO
•
•
xxx
xx
I
I
Pre-
Early
Late
Puva
Puva
Puva
Group I G r o u p II
x
MEDIUM SMALL NONE
OO Oe
xxx
331
xx
L
I
x •
Fig. 2. Mucin deposition in groups I and II. (pre-, early, late PU VA), there were no significant statistical differences. Post-PUVA all four patients (group III) had increased melanocytes; three of four had increased melanin content, with one showing a decrease. Dermal changes Elastin (Table HI), Elastic tissue was generally normal in all groups. There was some fragmentation early in PUVA. The fragmentation of elastic tissue was evaluated after a study of controls with nonpsoriatic inflammatory dermatoses, and there was no statistically significant difference (p > 0.6) between the groups and nonpsoriatic controls. Amyloid, No amyloid deposits were found in any patients before, during, or after PUVA therapy. All sections stained with Congo red were examined under polarized light. Collagen. No collagen abnormalities were found before, during, or after PUVA. Mucin. Group 1: Prior to therapy, nine patients sho wed no mucin deposits, and one patient showed small deposits. At 3 months, two patients showed no deposits, but eight patients showed small to large accumulated mucin deposits. After 2 years, nine patients continued to show mucin in small or medium amounts. These changes are statistically significant (p < 0.01) (Fig. 2). Group H: After 3 months of PUVA, five patients
xx
Pre-
•
l
I
Early
Post
Q
Group IliA x G r o u p IIIB •
Fig. 3. Mucin deposition in group HI. showed no mucin deposits, two had small, and one had large deposits. After 2 years, eight patients had small to large amounts of mucin deposition. These changes demonstrated a relationship that was statistically significant (p < 0.05) (Fig. 2). Group 111: There was no mucin staining before PUVA. All four patients showed deposition during PUVA (Fig. 3). After PUVA, themucin disappearedin all four patients. These samples were too small for statistical analysis. Immunofluoreseence a n d antinuelear antibody results. No antinuclear antibodies were found prior, during, or after PUVA in any of the patients, No biopsy was found to have specific staining o f IgG, IgM, IgA, C3, or C l q on direct immunofluorescence prior, during, or after PUVA in any of the patients. DISCUSSION A number of changes in the skin have been observed in patients receiving PUVA therapy. 4,5 These include epidermal dysplasia, ~ an increase in melanin pigmentation, 7,8 amyloid deposition, 9,~° mucin accumulation) and changes in the elastic tissue It in the dermis. Cox and Abel ~ found focal dystrophy of epidermal ceils in about half of their patients. These dystrophic cells were irregularly scattered in the epidermis and did not appear in
332
L e v i n et al
pretreatment controls. Single cells or clusters of ceils with large hyperchromatic nuclei were usually found in the basal cell layer, but could also be seen at higher levels. Some atypical nuclei were lobulated or double, with an irregular chromatin pattern. Isolated necrotic cells were also found. Gschnait and co-workers 7 recently found an increase in the amount of melanin in the epidermis in patients treated with PUVA. Melanin granules were found at all levels o f the epidermis, including the stratum comeum. Some melanocytes were large, with hyperchromatic and irregular nuclei. Similarly, Zelickson and co-workers 8 found an increase in the size of their melanosomes in patients treated with PUVA. They found melanocytes extending deep into the dermis, with alterations in internal organelles. Rosdahl and Swanbeck 1~ found no significant change in the number of DOPA-active melanocytes in psoriatic patients after P U V A therapy. T h e y concluded that P U V A treatment does not induce an increase in the formation of epidermal melanoeytes. Green and Cox 9 found focal amyloid deposits in involved and uninvolved skin of twelve of fiftytwo psoriatics treated with PUVA after 1 or 2 years o f therapy. These deposits were periodic a c i d - S c h i f f - p o s i t i v e and stained yellow with the V e r h o e f f - v a n Gieson stain. They were stained metachromatically by crystal violet and exhibited characteristic fluorescence after thioflavin T staining. Electron microscopic examination showed straight, nonbranching fibrils 60 to 100 A in diameter. These deposits were found in involved and uninvolved areas of skin. Hashimoto and Kurnakiri l° found colloid-amyloid bodies at the derrnoepidermal junction. They identified 60 to 70 straight, nonbranching, nonanastomosing filaments in these aggregations, qualifying them to be called arnyloid filaments as observed in primary Iocalized cutaneous amyloidosis. In addition, Congo red and crystal violet stains were occasionally positive in these colloid-amyloid bodies. Bergfeld s demonstrated an accumulation of acid rnucopoIysaccharides in the papillary dermis of psoriatics treated with PUVA. Zelickson and coworkers '1 found ultrastructural evidence for alterations in elastic tissue after just two weeks of
Journal of the American Academy of Dermatology
PUVA therapy. Early alterations were a decrease in the amount of elastin present, followed by disrupted microfibrils with inability to hold the fibers in the normal shape. Bergfeld5 was unable to demonstrate consistent changes in elastic tissues using light microscopy. Recently, Gschnait and co-workers 7 failed to demonstrate immunoglobulin deposition in the skin of 93% of patients treated with PUVA. Occasional staining with IgM in the horny layer, or IgG and IgM at the dermoepidermal junction, was not thought to be of significance. In a series by Bjellemp et al, '3 21% of patients developed positive ANAs during PUVA therapy• In a series of patients studied by Kubba,* 42% of patients developed a positive A N A . None of these patients had a positive anti-DNA or extractable nuclear antigen. Stern et al TM recently reported, in a multicenter cooperative 5-year prospective 'study of PUVA, that when first and last tests were compared, the incidence of positive ANAs was not significantly different. Pohl and Christophers 15 recently showed that 8-methoxypsoralen and UVA will cause a characteristic change of fibroblasts in tissue culture. These alterations include an increased number of polynuclear and hyperchromatic fibroblasts. Our study evaluated epidermal changes in PUVA-treated psoriatics and found no statistically significant increase in hyperkeratosis or atrophy. This may be due to the fact that most psoriatics treated with PUVA have previously used UVB and topical corticosteroid preparations. As such, the percentage of PUVA-treated psoriatics with hyperkeratosis and atrophy may be no greater than non-PUVA-treated psoriatic patients. More significantly, no dysplastic changes were found in the epidermis. We were unable to find dysplastic cells as had Cox and Abel G and Gschnait et al, r but we concur with Hashimoto et al 4 that epidermal keratinocytes remain normal. An increase in the number of melanocytes or the amount of melanin was not found with long-term PUVA therapy. This surprising finding may be accounted for again by previous UVB exposure of most PUVA patients. In addition, the amount of *Kubba R: Personal communication, 1980.
Volume 6 Number 3 March, 1982
melanin staining with hematoxylin and eosin may not correspond exactly to the pigmentation observed in an individual patient. Furthermore, skin color is determined by the number of stage I-IV melanosomes, distributed singly or in clumps, in epidermal melanocytes or their dendrites and in keratinocytes.16 These electron microscopic studies may or m a y not correlate with basal layer pigmentation seen with light microscopy. Our study agrees with Rosdahl and Swanbeck's n findings that melanocytes are not increased after PUVA therapy. The changes in elastic tissue were not consistent. We are impressed with the variability of the normal controls, as well as the study patients. Our findings probably point to the insensibility o f the staining technic, rather than the lack of PUVAinduced changes. No deposition o f amyloid was observed with the Congo red stain during PUVA therapy. More patients will need to be evaluated with the electron microscope and the Congo red method to determine whether this is a reliable method for detecting amyloid in PUVA-treated patients. None of our patients developed antinuclear antibodies while on P U V A therapy. This is contrary to a number o f other reports 12'1z but agrees with the findings of Stern et aI.14 Perhaps the discrepancy can be explained b y the careful pre-PUVA screening for positive A N A tests done in our patients. We were unable to find any specific deposits o f immunoglobulins or complement in our PUVAtreated patients, thus confirming the works of Gschnait and co-workers. 7 Finally, the deposition o f acid mucopolysaccarides seems to be correlated with the length of P U V A therapy. The mucin may be a product o f altered fibroblasts induced by P U V A therapy, 14 or it may be secreted by fibroblasts during the repair o f PUVA-induced dermal changes. Although only four patients had been off P U V A for a significant period of time, their mucin changes returned to normal after P U V A was stopped. This suggests that the deposition of acid mucopolysaccarides in the dermis due to P U V A therapy is reversible.
lmmunofluorescent and ANA findings in PUVA
333
REFERENCES
1. Pa~ish JA, Fitzpatrick TB, Tannenbaum L, et ak Photochemotherapy of psoriasis with oral methoxsalen and long wave ultraviolet light. N Engl J Med 291: I2071212, 1974. 2. Roenigk HH Jr, et al: Photochemotherapy for psoriasis--a clinical cooperative study of PUVA-48 and PUVA-64. Arch Dermatol 115:576-579, t979. 3. MelskiJW, Tanenbaum L, Parrish JA, et al: Oral methoxsalen photochemotherapy for the treatment of psoriasis: A cooperative clinical trial. J Invest Dermatol 68:328-335, 1977. 4. Hashimoto K, Kohda H, Kurnakiri M, et al: PsoralenUVA treated psoriatic lesions. Arch Derrnatol 114:711722, 1978. 5. Bergfeld WF: Histopathologic changes in skin after photochemotherapy. Cutis 20:504-507, 1977. 6. Cox A J, Abel EA: Epidermal dystrophy. Arch Dermatol 115:567-570, 1979. 7. Gschnait F, Wolff K, Honigsmaan H, et al: Long term photochemotherapy: Histopathological and immunofluorescence observations in 243 patients. Br J Dermatol 103: 11-22, 1980. 8. Zelickson AS, Mottaz JH, Muller SA: Melanocyte changes following PUVA therapy. J AM ACADDERMATOL 1:422-430, t979. 9. Green I, Cox AJ: Amyloid deposition after psoriasis therapy with psoralen and long-wave ultraviolet light. Arch Dermatol 115:1200-1202, 1979. i0. Hashimoto K, Kumakiri M: Colloid-amyloid bodies in PUVA-treated human psoriatic patients. J Invest Dermatol 72:70-79, 1979. 11. Zelickson AS, Mottaz JH, Zelickson BD, et at: Elastic tissue changes in skin following PUVA therapy. J AM ACM3 D~R~ATOL3: 186-192, 1980. 12. Rosdahl IK, Swanbeck G: Effects of PUVA on the epidermal rnelanocyte population in psoriatic patients. Acta Derm Venereol (Stockh) 60:21-26, 1980. 13. Bjellerup M, Bruze M, Forsgren A, et al: Antinuclear antibodies during PUVA therapy. Acta Derm Venereol (Stockh) 59:73-75, 1979. 14. Stem RS, Morison WL, Thibodeau LA, et al: Antinuclear antibodies and oral methoxsalen photochemotherapy (PUVA) for psoriasis. Arch Dermatol 115:13201324, 1979. 15. Pohl J, Christophers E: Photoinactivation and recovery in skin fibroblasts after formation of mono- and bifunctional adduets by furocoumarins-plus-UVA. J Invest DermatoI 75:306-3t0, 1980. 16. Fitzpatrick TB, Szabo G, Makoto S, Quevedo WC: Biology of the melanin pigmentary system, bz Fitzpatrick TB, et al, editor: Dermatology in general medicine. New York, 1979, McGraw-Hill Book Co., p. 145.
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Histologic, immunofluorescent, and antinuclear antibody findings in PUVA-treated patients Donald L. Levin, M.D., Henry H. Roenigk, Jr., M . D . , William A. Caro, M . D . , and Mary Lyons, M.D. Chicago, IL Twenty-two patients with psoriasis were treated with PUVA therapy for over two years. Skin biopsies from sun-exposed skin were evaluated before, during, and after PUVA therapy. No statistically significant epidermal changes were found when looking for hyperkeratosis, atrophy, melanin, or melanocyte nmnbers. No dyskeratosis was found. Dermal changes, including alterations in elastic tissue and collagen, were not statistically significant. Amyloid deposits were absent. The amount of mucin deposition was found to correlate with the length of PUVA therapy. In four patients, the mucin disappeared after withdrawal of PUVA, suggesting that this change is reversible. (J AM ACAD DERMATOL6:328-333, 1982.)
Photochemotherapy (PUVA), utilizing 8-methoxypsoralen (P) and long-wavelength ultraviolet light (UVA), has been used in the treatment of psoriasis since 1974.' It is an effective form of therapy" that is readily accepted by patients. The side effects of PUVA are well known 3 and include erythema, pruritus, nausea, formation of cutaneous tumors, and the possible development of cataracts. This prospective study will review the histopathologic findings in PUVA-treated patients and will present data on the direct immunofluorescent and antinuclear antibody (ANA) values in a series of patients with psoriasis treated with PUVA therapy. METHODS Twenty-two patients with psoriasis were randomly selected from the PUVA Center at Northwestern Uni-
From the Department of Dermatology, Northwestern University Medical School. Reprint requests to: Dr. Henry H. Roenigk, Jr., Northwestern University Medical School, Department of Dermatology, 303 East Chicago Ave., Chicago, IL 60611.
328
versity. All patients had psoriasis involving greater than 30% of the body surface area. All patients had been treated previously with topical steroids and ultraviolet B (UVB) therapy. Except for the four patients who discontinued treatment because of remission of their psoriasis, all patients continued to use topical corticosteroids in order to minimize their exposure to PUVA. The frequency of maintenance therapy was determined according to disease activity, in order to maintain clinical remission. Most patients on maintenance received treatments two to four times per month. 8-Methoxypsoralen (8-MOP) was ingested in doses of 0.5 to 0.6 mg/kg, according to weight and skin type, 2 hours before PUVA therapy. Patients were started at 1.0 joule/cm2; the dosage was increased until clearing of psoriasis was evident, according to standard methods. 2'3 Plateau method of giving PUVA 2 was used to minimize exposure to UVA. Four-millimeter skin biopsies for light microscopy and immunofluorescence were obtained from the upper arm (noninvolved, sun-exposed). Sections were stained with hematoxylin and eosin, iron, Congo red, and acid orcein-Giemsa according to standard procedures. Sections were examined without clinical information and without known order of sequence.
0190-9622/82/030328+06500.60/0 © 1982 Am Acad Dermatol
Volume 6 Number 3 March, 1982
hnmttnofluorescent and ANA findings in PUVA
329
T a b l e I. Patient population s u m m a r y , time of biopsy, age, and joules/cm 2 Biopsies (months after starting PUVA) Group
3 months
17-24 months
Age ± S.D.
Total joules/ cm z ± S.D,
x
x
x
x
x x
x x
x
x
47.6 ___ 14.8 50.1 ~ 13.9 35 59
2,219 _ 968 2,085 + 925 595 624
Number of patients
Pre-
I
10
II IIIA
8 2
IIIB
2
T a b l e II. E p i d e r m a l changes before, during, and after P U V A therapy
Hyperkeratosis Present Absent Atrophy Present Absent Melanocytes Decreased Normal Increased Melanin Decreased Normal Increased
Early
PrePUVA
PUVA
Late PUVA
PostPUVA
11 1
19 3
18 0
3 1
8 4
9 13
14 4
2 2
1 4 7
2 9 11
0 7 11
0 0 4
1 5 6
1 12 9
0 8 10
1 0 3
Biopsies for immunofluorescence were snap-frozen in liquid nitrogen, and specimens were stained with fluorescein-conjugated IgG, IgM, C3, and Clq, according to standard procedures. Serum ANA test was performed at the time of each skin biopsy. PATIENTS The psoriasis patients were divided into three groups (Table I). Groups I and II received continuous PUVA therapy, whereas group III went into remission with initial therapy; therefore, no further PUVA was given. Group I: Biopsy examination was done on ten patients (mean age, 47.6 ___ 14.8 years) before PUVA was initiated, 3 months later, and 2 years after PUVA was begun. Mean total UVA dosage was 2,219 ___968 joules/cm ~(mean +__S.D.). Group H: Biopsy examination was done on eight patients (mean age, 50.1 ___ 13.9 years) 3 months after PUVA therapy was initiated, and 2 years after PUVA therapy was begun. These patients did
not receive a pretreatment biopsy. Mean total UVA dosage was 2,085 + 925 joules/era 2 (mean ___ S.D.). Group IliA: Biopsies were done on two patients (average age, 35) prior to initiation of PUVA therapy, and 3 months after therapy was initiated. Due to remission at 6 months, PUVA therapy was terminated. Neither patient received PUVA therapy in the ensuing 18 months. Repeat skin biopsy was performed at 18 months. The average total dosage was 595 joules/era ~. Group IIIB: Biopsies were done on two additional patients (average age, 59) 3 months after initiation of therapy. These patients were treated until remission 4 months later and then were withdrawn from therapy. Biopsy examination was repeated 17 months after PUVA therapy ceased. The average total dosage was 624 joules/cm ~-.
330
Journal of the American Academy of Dermatology
L e v i n et al
Fig. 1. Epidermal changes showing hyperkeratosis, atrophy, increased melanocytes, and melanin content present in most biopsies. (Hematoxylin-eosin stain; x 400.)
Table III. Elastic tissue changes I -
Normal Decreased Fragmented Total
PrePUVA
Early PUVA
PUVA
PostPUVA
9 1 2 12
17
15
3
5 22
3 18
1 4
HISTOPATHOLOGY RESULTS
Epidermal changes Hyperkeratosis (Table II). Hyperkeratosis was present in the vast majority of biopsies examined at all stages of P U V A therapy. These changes were not statistically significant (p > 0.3) in any group (Fig. 1). A t r o p h y (Table II). Atrophy was present before PUVA, and late PUVA biopsies showed some increase in the number of patients with atrophy. In comparing all groups, there were no
Late
statistically significant differences (p > 0.1) between groups (Fig. 1). Dyskeratosis. We observed no evidence of dyskeratosis in any section studied at any time before, during, or after PUVA therapy (Fig. 1).
Basal layer melanocytes and melanin content (Table II). There were fewer melanocytes and less melanin in pretreatment biopsies than in early and late PUVA biopsies. There was a trend toward increased melanocytes and melanin content in early and late PUVA, but, comparing all groups
Volume 6 Number 3 March, 1982
hnrnunofluorescent and ANA findings in PUVA
MUCIN
MUCIN
LARGE
LARGE
MEDIUM
X
XX
Xx
SMALL
XX
xx
xx
•
xxx
NONE
•
•
x
•
xx
oe
xx
gO
•
•
xxx
xx
I
I
Pre-
Early
Late
Puva
Puva
Puva
Group I G r o u p II
x
MEDIUM SMALL NONE
OO Oe
xxx
331
xx
L
I
x •
Fig. 2. Mucin deposition in groups I and II. (pre-, early, late PU VA), there were no significant statistical differences. Post-PUVA all four patients (group III) had increased melanocytes; three of four had increased melanin content, with one showing a decrease. Dermal changes Elastin (Table HI), Elastic tissue was generally normal in all groups. There was some fragmentation early in PUVA. The fragmentation of elastic tissue was evaluated after a study of controls with nonpsoriatic inflammatory dermatoses, and there was no statistically significant difference (p > 0.6) between the groups and nonpsoriatic controls. Amyloid, No amyloid deposits were found in any patients before, during, or after PUVA therapy. All sections stained with Congo red were examined under polarized light. Collagen. No collagen abnormalities were found before, during, or after PUVA. Mucin. Group 1: Prior to therapy, nine patients sho wed no mucin deposits, and one patient showed small deposits. At 3 months, two patients showed no deposits, but eight patients showed small to large accumulated mucin deposits. After 2 years, nine patients continued to show mucin in small or medium amounts. These changes are statistically significant (p < 0.01) (Fig. 2). Group H: After 3 months of PUVA, five patients
xx
Pre-
•
l
I
Early
Post
Q
Group IliA x G r o u p IIIB •
Fig. 3. Mucin deposition in group HI. showed no mucin deposits, two had small, and one had large deposits. After 2 years, eight patients had small to large amounts of mucin deposition. These changes demonstrated a relationship that was statistically significant (p < 0.05) (Fig. 2). Group 111: There was no mucin staining before PUVA. All four patients showed deposition during PUVA (Fig. 3). After PUVA, themucin disappearedin all four patients. These samples were too small for statistical analysis. Immunofluoreseence a n d antinuelear antibody results. No antinuclear antibodies were found prior, during, or after PUVA in any of the patients, No biopsy was found to have specific staining o f IgG, IgM, IgA, C3, or C l q on direct immunofluorescence prior, during, or after PUVA in any of the patients. DISCUSSION A number of changes in the skin have been observed in patients receiving PUVA therapy. 4,5 These include epidermal dysplasia, ~ an increase in melanin pigmentation, 7,8 amyloid deposition, 9,~° mucin accumulation) and changes in the elastic tissue It in the dermis. Cox and Abel ~ found focal dystrophy of epidermal ceils in about half of their patients. These dystrophic cells were irregularly scattered in the epidermis and did not appear in
332
L e v i n et al
pretreatment controls. Single cells or clusters of ceils with large hyperchromatic nuclei were usually found in the basal cell layer, but could also be seen at higher levels. Some atypical nuclei were lobulated or double, with an irregular chromatin pattern. Isolated necrotic cells were also found. Gschnait and co-workers 7 recently found an increase in the amount of melanin in the epidermis in patients treated with PUVA. Melanin granules were found at all levels o f the epidermis, including the stratum comeum. Some melanocytes were large, with hyperchromatic and irregular nuclei. Similarly, Zelickson and co-workers 8 found an increase in the size of their melanosomes in patients treated with PUVA. They found melanocytes extending deep into the dermis, with alterations in internal organelles. Rosdahl and Swanbeck 1~ found no significant change in the number of DOPA-active melanocytes in psoriatic patients after P U V A therapy. T h e y concluded that P U V A treatment does not induce an increase in the formation of epidermal melanoeytes. Green and Cox 9 found focal amyloid deposits in involved and uninvolved skin of twelve of fiftytwo psoriatics treated with PUVA after 1 or 2 years o f therapy. These deposits were periodic a c i d - S c h i f f - p o s i t i v e and stained yellow with the V e r h o e f f - v a n Gieson stain. They were stained metachromatically by crystal violet and exhibited characteristic fluorescence after thioflavin T staining. Electron microscopic examination showed straight, nonbranching fibrils 60 to 100 A in diameter. These deposits were found in involved and uninvolved areas of skin. Hashimoto and Kurnakiri l° found colloid-amyloid bodies at the derrnoepidermal junction. They identified 60 to 70 straight, nonbranching, nonanastomosing filaments in these aggregations, qualifying them to be called arnyloid filaments as observed in primary Iocalized cutaneous amyloidosis. In addition, Congo red and crystal violet stains were occasionally positive in these colloid-amyloid bodies. Bergfeld s demonstrated an accumulation of acid rnucopoIysaccharides in the papillary dermis of psoriatics treated with PUVA. Zelickson and coworkers '1 found ultrastructural evidence for alterations in elastic tissue after just two weeks of
Journal of the American Academy of Dermatology
PUVA therapy. Early alterations were a decrease in the amount of elastin present, followed by disrupted microfibrils with inability to hold the fibers in the normal shape. Bergfeld5 was unable to demonstrate consistent changes in elastic tissues using light microscopy. Recently, Gschnait and co-workers 7 failed to demonstrate immunoglobulin deposition in the skin of 93% of patients treated with PUVA. Occasional staining with IgM in the horny layer, or IgG and IgM at the dermoepidermal junction, was not thought to be of significance. In a series by Bjellemp et al, '3 21% of patients developed positive ANAs during PUVA therapy• In a series of patients studied by Kubba,* 42% of patients developed a positive A N A . None of these patients had a positive anti-DNA or extractable nuclear antigen. Stern et al TM recently reported, in a multicenter cooperative 5-year prospective 'study of PUVA, that when first and last tests were compared, the incidence of positive ANAs was not significantly different. Pohl and Christophers 15 recently showed that 8-methoxypsoralen and UVA will cause a characteristic change of fibroblasts in tissue culture. These alterations include an increased number of polynuclear and hyperchromatic fibroblasts. Our study evaluated epidermal changes in PUVA-treated psoriatics and found no statistically significant increase in hyperkeratosis or atrophy. This may be due to the fact that most psoriatics treated with PUVA have previously used UVB and topical corticosteroid preparations. As such, the percentage of PUVA-treated psoriatics with hyperkeratosis and atrophy may be no greater than non-PUVA-treated psoriatic patients. More significantly, no dysplastic changes were found in the epidermis. We were unable to find dysplastic cells as had Cox and Abel G and Gschnait et al, r but we concur with Hashimoto et al 4 that epidermal keratinocytes remain normal. An increase in the number of melanocytes or the amount of melanin was not found with long-term PUVA therapy. This surprising finding may be accounted for again by previous UVB exposure of most PUVA patients. In addition, the amount of *Kubba R: Personal communication, 1980.
Volume 6 Number 3 March, 1982
melanin staining with hematoxylin and eosin may not correspond exactly to the pigmentation observed in an individual patient. Furthermore, skin color is determined by the number of stage I-IV melanosomes, distributed singly or in clumps, in epidermal melanocytes or their dendrites and in keratinocytes.16 These electron microscopic studies may or m a y not correlate with basal layer pigmentation seen with light microscopy. Our study agrees with Rosdahl and Swanbeck's n findings that melanocytes are not increased after PUVA therapy. The changes in elastic tissue were not consistent. We are impressed with the variability of the normal controls, as well as the study patients. Our findings probably point to the insensibility o f the staining technic, rather than the lack of PUVAinduced changes. No deposition o f amyloid was observed with the Congo red stain during PUVA therapy. More patients will need to be evaluated with the electron microscope and the Congo red method to determine whether this is a reliable method for detecting amyloid in PUVA-treated patients. None of our patients developed antinuclear antibodies while on P U V A therapy. This is contrary to a number o f other reports 12'1z but agrees with the findings of Stern et aI.14 Perhaps the discrepancy can be explained b y the careful pre-PUVA screening for positive A N A tests done in our patients. We were unable to find any specific deposits o f immunoglobulins or complement in our PUVAtreated patients, thus confirming the works of Gschnait and co-workers. 7 Finally, the deposition o f acid mucopolysaccarides seems to be correlated with the length of P U V A therapy. The mucin may be a product o f altered fibroblasts induced by P U V A therapy, 14 or it may be secreted by fibroblasts during the repair o f PUVA-induced dermal changes. Although only four patients had been off P U V A for a significant period of time, their mucin changes returned to normal after P U V A was stopped. This suggests that the deposition of acid mucopolysaccarides in the dermis due to P U V A therapy is reversible.
lmmunofluorescent and ANA findings in PUVA
333
REFERENCES
1. Pa~ish JA, Fitzpatrick TB, Tannenbaum L, et ak Photochemotherapy of psoriasis with oral methoxsalen and long wave ultraviolet light. N Engl J Med 291: I2071212, 1974. 2. Roenigk HH Jr, et al: Photochemotherapy for psoriasis--a clinical cooperative study of PUVA-48 and PUVA-64. Arch Dermatol 115:576-579, t979. 3. MelskiJW, Tanenbaum L, Parrish JA, et al: Oral methoxsalen photochemotherapy for the treatment of psoriasis: A cooperative clinical trial. J Invest Dermatol 68:328-335, 1977. 4. Hashimoto K, Kohda H, Kurnakiri M, et al: PsoralenUVA treated psoriatic lesions. Arch Derrnatol 114:711722, 1978. 5. Bergfeld WF: Histopathologic changes in skin after photochemotherapy. Cutis 20:504-507, 1977. 6. Cox A J, Abel EA: Epidermal dystrophy. Arch Dermatol 115:567-570, 1979. 7. Gschnait F, Wolff K, Honigsmaan H, et al: Long term photochemotherapy: Histopathological and immunofluorescence observations in 243 patients. Br J Dermatol 103: 11-22, 1980. 8. Zelickson AS, Mottaz JH, Muller SA: Melanocyte changes following PUVA therapy. J AM ACADDERMATOL 1:422-430, t979. 9. Green I, Cox AJ: Amyloid deposition after psoriasis therapy with psoralen and long-wave ultraviolet light. Arch Dermatol 115:1200-1202, 1979. i0. Hashimoto K, Kumakiri M: Colloid-amyloid bodies in PUVA-treated human psoriatic patients. J Invest Dermatol 72:70-79, 1979. 11. Zelickson AS, Mottaz JH, Zelickson BD, et at: Elastic tissue changes in skin following PUVA therapy. J AM ACM3 D~R~ATOL3: 186-192, 1980. 12. Rosdahl IK, Swanbeck G: Effects of PUVA on the epidermal rnelanocyte population in psoriatic patients. Acta Derm Venereol (Stockh) 60:21-26, 1980. 13. Bjellerup M, Bruze M, Forsgren A, et al: Antinuclear antibodies during PUVA therapy. Acta Derm Venereol (Stockh) 59:73-75, 1979. 14. Stem RS, Morison WL, Thibodeau LA, et al: Antinuclear antibodies and oral methoxsalen photochemotherapy (PUVA) for psoriasis. Arch Dermatol 115:13201324, 1979. 15. Pohl J, Christophers E: Photoinactivation and recovery in skin fibroblasts after formation of mono- and bifunctional adduets by furocoumarins-plus-UVA. J Invest DermatoI 75:306-3t0, 1980. 16. Fitzpatrick TB, Szabo G, Makoto S, Quevedo WC: Biology of the melanin pigmentary system, bz Fitzpatrick TB, et al, editor: Dermatology in general medicine. New York, 1979, McGraw-Hill Book Co., p. 145.