British Journal of Dermatology (1977) 97* 629.
Pityriasis lichenoides—an immune complex disease ROGER CLAYTON, GERALD HAFFENDEN, ANTHONY DU VIVIER, JILL BURTON AND JAMES MOWBRAY Departments of Dermatology, Histopathology and Experimental Pathology, St Mary's Hospital, London W2 Accepted for publication 10 July 1977
SUMMARY
Circulating immune complexes have been detected in patients with pityriasis lichenoides during disease activity when IgM and C3 have been observed in dermal vessels on direct immunofluorescence of fresh lesions. This imphes that pityriasis hchenoides is an immune complex disorder and that deposited complexes play a part in the pathogenesis ofthe condition. There is a characteristic pattern of immunofluorescence which may be a diagnostic aid.
Pityriasis lichenoides (PL) is a skin disorder occurring mainly in adolescents and young adults. Occasionally fever and constitutional symptoms precede the eruption which favours the trunk and proximal limbs. Lesions last 2-12 weeks. In the acute form an initial purpuric papule 2-8 mm across vesiculates, with haemorrhagic necrosis and scar formation. Chronic lesions develop a centrally adherent scale which is shed prior to resolution. The course is variable; fresh crops of lesions may cease after a few weeks and many cases clear within 6 months. There may be intermittent recurrence or continuous activity over a period of years. Microscopy of the lesions shows an upper dermal mononuclear inflammatory cell infiltrate which is predominantly perivascular and frequently invades the epidermis. Autoradiographic and histochemical studies (Black & Marks, 1972) suggest minimal vascular damage. We have investigated a group of 16 patients with PL who satisfy previous diagnostic criteria (Marks et ah, 1972) and report new immunofluorescent (IF) and serological findings. PATIENTS AND METHODS
Nine female and 7 male patients aged 9-79 years were studied. The duration of disease ranged from 9 months to 22 years. Two patients had the acute form ofthe disease with blisters. One has rheumatoid arthritis (RA) and two others had symptoms of polyarthritis, necessitating phenylbutazone therapy, just prior to the onset ofthe eruption. Four millimetre punch skin biopsies of fresh papular, non-scaly, purpuric lesions were embedded in Tissue-Tek II OCT Compound (Lab-Tek, Miles) and frozen in liquid nitrogen. Five-micron sections were stained with optimally diluted conjugates of sheep anti-human IgG, IgM and IgA (Wellcome) and rabbit anti-human C3 (Dakopatts). The sections were examined for direct immunofluorescence 629
630
R.Clayton et al.
(IF) in a Leitz Dialux microscope with Ploem incident illumination. Tbe filter system comprised: exciter 2 x KP 490, dichroic mirror TK 510, suppression K515 and edge filter GG 475. In 3 patients uninvolved skin within an area where lesions were forming was examined. The normal skin of 16 healthy control subjects without a rash was also studied by the same technique. Blood samples were obtained from patients both during disease activity, when crops of fresh lesions were appearing, and during quiescence. Separated serum was stored at -70 C until analysed. Detection of soluble immune complexes
The assay used the precipitation of soluble immune complexes from serum by the addition of polyethylene glycol (PEG) (PEGj molecular weight, 6000; Hopkin & Williams) (Nydegger et al., 1974). The amount of Ciq and IgG in the precipitate was measured by single radial immunodifFusion (SRID). These values were compared with those obtained in this test from a pool of sera from 25 healthy individuals. 01 ml of a solution of 12*^;, PEG in isotonic saline containing 60 mM ethyiene diamine tetra-acetic acid (EDTA), pH 76, was added to 05 ml of the test serum, either used fresh or stored at -70 C, in a polystyrene tube. The solutions were mixed and left at -| 4 C for 18 h. The solutions were mixed again before centrifuging at 1000 g for 20 min at -f 4^^ C. The supematants were decanted by pouring and kept at + 4 C. The precipitates were washed once in 2 ml of 2'^i PBG in isotonic saline containing 10 nM EDTA, pH 7-6. The tubes were drained briefiy and the precipitates dissolved in 0-15 M saline. The solution was mixed and left for 30 min before assaying IgG and Ciq. The concentrations of Ciq and IgG in the pellet and Ciq in the starting serum and supernatant were determined by SRID in i ml thick gels of i",, agarose in 40 mM EDTA buffered isotonic saline, pH 8-6. The gel also usually contained 10";, antiserum. Ten fil samples were placed in 4 mm diameter wells and the ring diameters measured after incubation at 37 C overnight. The amounts of antigen were calculated from the values obtained for the standards included in each plate. The antisera used were raised in rabbits by immunization with purified components and standardized by comparison with known reference antibodies. Each batch of sera contained at least 2 control sera. The values obtained from these sera have provided the confidence limits for the test. Soluble immime complexes are deemed to be present if the value for precipitated Ciq is > 5 i ' 5 % of the total Ciq (mean 34'^'n, s.d. 17-5",,); or if the value for precipitated IgG is •o69O"n (mean 0485';,,, s.d. O2O5",j). Measurement of the supernatant Ciq allows detection of complexes which bind all the available Ciq, but otherwise they give an indication of the recovery of Ciq in the experiment. Serum antistreptolysin O (ASO) titres and toxoplasma complement fixation titres (dye method) were also determined. RESULTS
In the majority of biopsies of fresh lesions, granular deposits of IgM and C3 were found in the walls of dermal papillary vessels and also along the dermal-epidermal junction (D-EJ) (Fig. i). In general the IF was not as bright or extensive as that which we have seen in the vessels or along the D-EJ of other cutaneous disorders. IgG and IgA were not found in any of the biopsies. Table i shows that IgM and C3 were not always detected together, nor were they always present both in the vessels and at D-EJ. The ratio of Ciq to IgG in the precipitated complexes was high implying the presence also of IgM antibodies. Fig. 2 shows the frequency of detected complexes during clinical disease activity compared with that of healthy subjects. Complexes were found only once on 5 occasions when disease was
Pityriasis lichenoides
631
ni
u
U
R.Clayton et al.
632
TABLE 1
Patienr I
2
3
4
Date
Disease activity
I
I2-I 26-2
N.D.
26-4
UI
17 99 1410
N.D. N.D.
11-2
I I
267
N.D.
27-2 21-6 197
I I
153
I UI
N.D. N.D. I
1510
6 7
153 17-5
4-5
8
175
9
17-5 266 28
8-10 10
10-6
II
267 2-8
8-10 12
307
13
138
14
25-10
15
n i l
16
4-
304 2110 21-6 8-10
15 II
D-EJ IF Igm C3
—
—
-
-
— —
— —
J-
— 44"
-h
+
I
444-
I I
—
I
Vessel IF IgM C3
Complexes
-i-
N.D. I
4-5
5
In,'uninvolved skin (I/UI)
N.D. —
N.D 44-
44-
N.D N.D
+ .
N.D 4N.D
+ 4-
+ -
4N.D
4-
N.D. N.D. I I
— —
-
— —
—
4-
N.D. N.D. N.D. I I I I I I I I I UI N.D. = not done.
4-
—
4— 4-
— _
4— _
—
44-
N.D.
Pityriasis Hchenoides
633
TqM preseni ( 7 pQiients)
IOO CoTipleies detected
iqM absent 111 patients)
IOC
56%
100
Complexes 7 i % deteded
Heollhy . controls
Fig. 2.
Fig 3
FIGURE 2. Detection of complexes in patients during active disease (25 estimations in the 16 patients). FIGURE 3. Correlation of detected complexes with IgM in biopsies.
inactive; this was in the patient with active RA. Fig. 3 shows that there is a good correlation between the presence of detectable circulating complexes and biopsies positive for IgM. In the 3 biopsies of uninvolved skin IgM and C3 were found in only one. In the 16 biopsies of normal skin of healthy subjects faint IF was seen in only one—IgM and C3 in superficial dermal vessels. The ASO and toxoplasma antibody titres were within normal limits in each patient. DISCUSSION
The presence of circulating immune complexes during disease activity when IgM and C3 IF is observed in dermal vessels of fresh lesions suggests that the complexes are deposited in the vessels and play a role in the pathogenesis of PL. The IgM and C3 deposits at the D-EJ and in vessel walls that we have demonstrated appear to be characteristic ofthe condition and may therefore aid diagnosis. There is a surprising lack of polymorphs in the lesion of PL considering the frequency of C3 detection. But in support of our contention that lesions are caused by deposition of complexes, the uninvolved skin in 2 of 3 patients showed no IgM or C3. The nature of complex immunoglobulin and antigen both contribute to the clinical and histological features of immune complex disease. The deposition only of IgM/C3 complexes may be responsible for the rare clinical manifestations of PL in which the histology ofthe lesion is often not diagnostic. One of our patients has RA and two others had an initial polyarthritis. It is of interest that another patient with PL and RA has been described (Muller & Schulze, 1971). Ciq binding serum complexes are more common in rheumatoid patients with extra-articular vasculitic disease than in those with arthritis alone (Zubler et al., 1976). Vessel IgM and C3 have been found in both involved and uninvolved skin of rheumatoid patients with cutaneous vasculitis (Schroeter ei al, 1976). This implies that, as in PL, the deposited complexes are predominantly of the IgM class and play a part in the causation ofthe skin lesions. PL may be part of a disease spectrum produced by IgM complexes. Piamphongsant (1974) has shown that some patients with PL respond to tctracycline treatment. He suggested that the disease may be a hypersensitivity to a focus of infection. PL patients with a raised toxoplasma antibody titre have responded to pyrimethamine (Zlatkov & Andreev, 1972) but none of our patients or those of Ceilley & Zuehlke (1976) had an elevated titre. Also there was no evidence of streptococcal infection in our patients in that their ASO titres were not raised. A rational treatment regime of PL must relate to the antigenic component ofthe complex.
634
R.Clayton et al. ACKNOWLEDGMENTS
We thank Drs M.Feiwel, L.Fry, D.S.Wilkinson (Wycombe General Hospital) and Professor M. Greaves (St John's Hospital) for permission to study their patients. Mr William Russell took and developed the photographs. This work was aided in part by grants to one of us (JM) from the Medical Research Council.
REFERENCES BLACK, M.M. & MARKS, R. (1972) The inflammatory reaction in pityriasis lichenoides. British Journal of Dermatology, 87, 533. CEILLEY, R.I. &ZUEHLKE, R.L. (1976) Pityriasis Hchenoides and toxoplasmosis. Archives of Dermatology, 112,557. MARKS, R., BLACK, M.M. & WILSON JONES, E. (1972) Pityriasis lichenoides—a reappraisal. British Journal of Dermatology, 86, 215. MULLER. S.A. & SCHULZE, T.W., JR (1971) Alucha-Habermann disease mistaken for reticulum cell sarcoma. Archives of Dermatology, 103, 423. NYDEGGER, U.E., LAMBERT, P.H., GERBER, H . & MIESCHER, P.A. (1974) Circulating immune complexes in the
serum in systemic lupus erythematosus and in carriers of hepatitis B antigen. Quantitation by binding to radiolabelled Ciq. Journal of Clinical Investigations, 54, 297. PlAMPHONGSANT, T. (1974) Tetracycline for the treatment of pityriasis lichenoides. British Journal of Dermatology, 91, 319ScHROETER, A.L., CoNN, D.L. & JoRDON, R.E. (1976) ImmunoglobuHn and complement deposition in the skin of rheumatoid arthritis and systemic lupus erythematous patients. Annals of Rheumatic Diseases, 35, 321. ZLATKOV, N . B . & ANDREEV, V . C (1972) Toxoplasmosis and pityriasis lichenoides. British Journal of Dermatology, 87,114. ZuBLER, R.H., NYDEGGER, U . , PERRIN, L.H., FEHR, K . , MCCORMICK, J., LAMBERT, R.H. & MIESCHER, P.A.
(1976) Circulating and intra-articular immune complexes in patients with rheumatoid arthritis. JowrHtj/ of Clinical Investigations, 57, 1308.
Pityriasis lichenoides—an immune complex disease ROGER CLAYTON, GERALD HAFFENDEN, ANTHONY DU VIVIER, JILL BURTON AND JAMES MOWBRAY Departments of Dermatology, Histopathology and Experimental Pathology, St Mary's Hospital, London W2 Accepted for publication 10 July 1977
SUMMARY
Circulating immune complexes have been detected in patients with pityriasis lichenoides during disease activity when IgM and C3 have been observed in dermal vessels on direct immunofluorescence of fresh lesions. This imphes that pityriasis hchenoides is an immune complex disorder and that deposited complexes play a part in the pathogenesis ofthe condition. There is a characteristic pattern of immunofluorescence which may be a diagnostic aid.
Pityriasis lichenoides (PL) is a skin disorder occurring mainly in adolescents and young adults. Occasionally fever and constitutional symptoms precede the eruption which favours the trunk and proximal limbs. Lesions last 2-12 weeks. In the acute form an initial purpuric papule 2-8 mm across vesiculates, with haemorrhagic necrosis and scar formation. Chronic lesions develop a centrally adherent scale which is shed prior to resolution. The course is variable; fresh crops of lesions may cease after a few weeks and many cases clear within 6 months. There may be intermittent recurrence or continuous activity over a period of years. Microscopy of the lesions shows an upper dermal mononuclear inflammatory cell infiltrate which is predominantly perivascular and frequently invades the epidermis. Autoradiographic and histochemical studies (Black & Marks, 1972) suggest minimal vascular damage. We have investigated a group of 16 patients with PL who satisfy previous diagnostic criteria (Marks et ah, 1972) and report new immunofluorescent (IF) and serological findings. PATIENTS AND METHODS
Nine female and 7 male patients aged 9-79 years were studied. The duration of disease ranged from 9 months to 22 years. Two patients had the acute form ofthe disease with blisters. One has rheumatoid arthritis (RA) and two others had symptoms of polyarthritis, necessitating phenylbutazone therapy, just prior to the onset ofthe eruption. Four millimetre punch skin biopsies of fresh papular, non-scaly, purpuric lesions were embedded in Tissue-Tek II OCT Compound (Lab-Tek, Miles) and frozen in liquid nitrogen. Five-micron sections were stained with optimally diluted conjugates of sheep anti-human IgG, IgM and IgA (Wellcome) and rabbit anti-human C3 (Dakopatts). The sections were examined for direct immunofluorescence 629
630
R.Clayton et al.
(IF) in a Leitz Dialux microscope with Ploem incident illumination. Tbe filter system comprised: exciter 2 x KP 490, dichroic mirror TK 510, suppression K515 and edge filter GG 475. In 3 patients uninvolved skin within an area where lesions were forming was examined. The normal skin of 16 healthy control subjects without a rash was also studied by the same technique. Blood samples were obtained from patients both during disease activity, when crops of fresh lesions were appearing, and during quiescence. Separated serum was stored at -70 C until analysed. Detection of soluble immune complexes
The assay used the precipitation of soluble immune complexes from serum by the addition of polyethylene glycol (PEG) (PEGj molecular weight, 6000; Hopkin & Williams) (Nydegger et al., 1974). The amount of Ciq and IgG in the precipitate was measured by single radial immunodifFusion (SRID). These values were compared with those obtained in this test from a pool of sera from 25 healthy individuals. 01 ml of a solution of 12*^;, PEG in isotonic saline containing 60 mM ethyiene diamine tetra-acetic acid (EDTA), pH 76, was added to 05 ml of the test serum, either used fresh or stored at -70 C, in a polystyrene tube. The solutions were mixed and left at -| 4 C for 18 h. The solutions were mixed again before centrifuging at 1000 g for 20 min at -f 4^^ C. The supematants were decanted by pouring and kept at + 4 C. The precipitates were washed once in 2 ml of 2'^i PBG in isotonic saline containing 10 nM EDTA, pH 7-6. The tubes were drained briefiy and the precipitates dissolved in 0-15 M saline. The solution was mixed and left for 30 min before assaying IgG and Ciq. The concentrations of Ciq and IgG in the pellet and Ciq in the starting serum and supernatant were determined by SRID in i ml thick gels of i",, agarose in 40 mM EDTA buffered isotonic saline, pH 8-6. The gel also usually contained 10";, antiserum. Ten fil samples were placed in 4 mm diameter wells and the ring diameters measured after incubation at 37 C overnight. The amounts of antigen were calculated from the values obtained for the standards included in each plate. The antisera used were raised in rabbits by immunization with purified components and standardized by comparison with known reference antibodies. Each batch of sera contained at least 2 control sera. The values obtained from these sera have provided the confidence limits for the test. Soluble immime complexes are deemed to be present if the value for precipitated Ciq is > 5 i ' 5 % of the total Ciq (mean 34'^'n, s.d. 17-5",,); or if the value for precipitated IgG is •o69O"n (mean 0485';,,, s.d. O2O5",j). Measurement of the supernatant Ciq allows detection of complexes which bind all the available Ciq, but otherwise they give an indication of the recovery of Ciq in the experiment. Serum antistreptolysin O (ASO) titres and toxoplasma complement fixation titres (dye method) were also determined. RESULTS
In the majority of biopsies of fresh lesions, granular deposits of IgM and C3 were found in the walls of dermal papillary vessels and also along the dermal-epidermal junction (D-EJ) (Fig. i). In general the IF was not as bright or extensive as that which we have seen in the vessels or along the D-EJ of other cutaneous disorders. IgG and IgA were not found in any of the biopsies. Table i shows that IgM and C3 were not always detected together, nor were they always present both in the vessels and at D-EJ. The ratio of Ciq to IgG in the precipitated complexes was high implying the presence also of IgM antibodies. Fig. 2 shows the frequency of detected complexes during clinical disease activity compared with that of healthy subjects. Complexes were found only once on 5 occasions when disease was
Pityriasis lichenoides
631
ni
u
U
R.Clayton et al.
632
TABLE 1
Patienr I
2
3
4
Date
Disease activity
I
I2-I 26-2
N.D.
26-4
UI
17 99 1410
N.D. N.D.
11-2
I I
267
N.D.
27-2 21-6 197
I I
153
I UI
N.D. N.D. I
1510
6 7
153 17-5
4-5
8
175
9
17-5 266 28
8-10 10
10-6
II
267 2-8
8-10 12
307
13
138
14
25-10
15
n i l
16
4-
304 2110 21-6 8-10
15 II
D-EJ IF Igm C3
—
—
-
-
— —
— —
J-
— 44"
-h
+
I
444-
I I
—
I
Vessel IF IgM C3
Complexes
-i-
N.D. I
4-5
5
In,'uninvolved skin (I/UI)
N.D. —
N.D 44-
44-
N.D N.D
+ .
N.D 4N.D
+ 4-
+ -
4N.D
4-
N.D. N.D. I I
— —
-
— —
—
4-
N.D. N.D. N.D. I I I I I I I I I UI N.D. = not done.
4-
—
4— 4-
— _
4— _
—
44-
N.D.
Pityriasis Hchenoides
633
TqM preseni ( 7 pQiients)
IOO CoTipleies detected
iqM absent 111 patients)
IOC
56%
100
Complexes 7 i % deteded
Heollhy . controls
Fig. 2.
Fig 3
FIGURE 2. Detection of complexes in patients during active disease (25 estimations in the 16 patients). FIGURE 3. Correlation of detected complexes with IgM in biopsies.
inactive; this was in the patient with active RA. Fig. 3 shows that there is a good correlation between the presence of detectable circulating complexes and biopsies positive for IgM. In the 3 biopsies of uninvolved skin IgM and C3 were found in only one. In the 16 biopsies of normal skin of healthy subjects faint IF was seen in only one—IgM and C3 in superficial dermal vessels. The ASO and toxoplasma antibody titres were within normal limits in each patient. DISCUSSION
The presence of circulating immune complexes during disease activity when IgM and C3 IF is observed in dermal vessels of fresh lesions suggests that the complexes are deposited in the vessels and play a role in the pathogenesis of PL. The IgM and C3 deposits at the D-EJ and in vessel walls that we have demonstrated appear to be characteristic ofthe condition and may therefore aid diagnosis. There is a surprising lack of polymorphs in the lesion of PL considering the frequency of C3 detection. But in support of our contention that lesions are caused by deposition of complexes, the uninvolved skin in 2 of 3 patients showed no IgM or C3. The nature of complex immunoglobulin and antigen both contribute to the clinical and histological features of immune complex disease. The deposition only of IgM/C3 complexes may be responsible for the rare clinical manifestations of PL in which the histology ofthe lesion is often not diagnostic. One of our patients has RA and two others had an initial polyarthritis. It is of interest that another patient with PL and RA has been described (Muller & Schulze, 1971). Ciq binding serum complexes are more common in rheumatoid patients with extra-articular vasculitic disease than in those with arthritis alone (Zubler et al., 1976). Vessel IgM and C3 have been found in both involved and uninvolved skin of rheumatoid patients with cutaneous vasculitis (Schroeter ei al, 1976). This implies that, as in PL, the deposited complexes are predominantly of the IgM class and play a part in the causation ofthe skin lesions. PL may be part of a disease spectrum produced by IgM complexes. Piamphongsant (1974) has shown that some patients with PL respond to tctracycline treatment. He suggested that the disease may be a hypersensitivity to a focus of infection. PL patients with a raised toxoplasma antibody titre have responded to pyrimethamine (Zlatkov & Andreev, 1972) but none of our patients or those of Ceilley & Zuehlke (1976) had an elevated titre. Also there was no evidence of streptococcal infection in our patients in that their ASO titres were not raised. A rational treatment regime of PL must relate to the antigenic component ofthe complex.
634
R.Clayton et al. ACKNOWLEDGMENTS
We thank Drs M.Feiwel, L.Fry, D.S.Wilkinson (Wycombe General Hospital) and Professor M. Greaves (St John's Hospital) for permission to study their patients. Mr William Russell took and developed the photographs. This work was aided in part by grants to one of us (JM) from the Medical Research Council.
REFERENCES BLACK, M.M. & MARKS, R. (1972) The inflammatory reaction in pityriasis lichenoides. British Journal of Dermatology, 87, 533. CEILLEY, R.I. &ZUEHLKE, R.L. (1976) Pityriasis Hchenoides and toxoplasmosis. Archives of Dermatology, 112,557. MARKS, R., BLACK, M.M. & WILSON JONES, E. (1972) Pityriasis lichenoides—a reappraisal. British Journal of Dermatology, 86, 215. MULLER. S.A. & SCHULZE, T.W., JR (1971) Alucha-Habermann disease mistaken for reticulum cell sarcoma. Archives of Dermatology, 103, 423. NYDEGGER, U.E., LAMBERT, P.H., GERBER, H . & MIESCHER, P.A. (1974) Circulating immune complexes in the
serum in systemic lupus erythematosus and in carriers of hepatitis B antigen. Quantitation by binding to radiolabelled Ciq. Journal of Clinical Investigations, 54, 297. PlAMPHONGSANT, T. (1974) Tetracycline for the treatment of pityriasis lichenoides. British Journal of Dermatology, 91, 319ScHROETER, A.L., CoNN, D.L. & JoRDON, R.E. (1976) ImmunoglobuHn and complement deposition in the skin of rheumatoid arthritis and systemic lupus erythematous patients. Annals of Rheumatic Diseases, 35, 321. ZLATKOV, N . B . & ANDREEV, V . C (1972) Toxoplasmosis and pityriasis lichenoides. British Journal of Dermatology, 87,114. ZuBLER, R.H., NYDEGGER, U . , PERRIN, L.H., FEHR, K . , MCCORMICK, J., LAMBERT, R.H. & MIESCHER, P.A.
(1976) Circulating and intra-articular immune complexes in patients with rheumatoid arthritis. JowrHtj/ of Clinical Investigations, 57, 1308.
