Supplement 1, Vol 8, Aust N Z J Med (1978), pp 87-89
The Value of lmmunofluorescence at the Dermal/Epidermal Junction in Connective Tissue Disorders D. R. Langlands and R. L. Dawkins
From the Departments of Rheumatology and Clinical Immunology, Royal Perth Hospital and Queen Elizabeth II Medical Centre, Perth, Western Australia
Since the pioneering work of Burnham and Fine' and Cormane2, immunofluorescent staining of the epidermalidermal junction has been used as a diagnostic aid for systemic lupus erythematosus (SLE). Recent reports, however, have suggested that junctional staining may be found in mixed connective tissue disease (MCTD)3and in rheumatoid arthritis (KA).s The latter finding is in marked contrast to previous studies5,6 , and the subsequent report by DeBoer and associate^.^ Over the last five years, we have had the opportunity to examine skin biopsies from patients with various connective tissue diseases. In this report we have attempted to determine the diagnostic specificity of junctional staining since biopsies have been obtained from patients with such diverse disorders as SLE, MCTD, KA, progressive systemic sclerosis (PSS), polymyositis (PM) and myasthenia gravis (MG).
Results
Results for the well-defined groups are tabulated in Table 1.,It is noteworthy that while eight of the 20 cases of SLE had positive results, these included five of the seven patients with significant renal disease. The single patient with a positive result in the RA group had only very weak fluorescence with the monospecific anti IgG conjugate. It may be relevant that this patient was on penicillamine therapy, and had ANF and other autoantibodies. TABLE 1 Dermallepidermal junctional fluorescence in connective tissue diseases*
Methods and Patients
Skin biopsies were taken for various indications. In SLE and in those patients in which this diagnosis was entertained, uninvolved skin was taken generally from the forearm. In cases with a suspicion of PM, the skin was obtained from the incision at the time of muscle biopsy. In patients with RA, the biopsy was usually taken because of a skin abnormality such as suspicion of a drug eruption or vasculitis. In the case of patients in which PSS was considered, and in many of those with MCTD, skin was often taken from the phalanges. linmunofluorescence
The specimen was embedded in Ames OCT compound, quick frozen in liquid nitrogen and stored at -70 'C until processed. Cryostat sections 6 1-1 in thickness were stained using fluorescein conjugated antisera to IgG, IgM, IgA and C3. Recent biopsies have also been assessed using C4 and Clq conjugates. Clinical Details Sixty patients were selected for retrospective review, so as to include cases with established diagnoses, as shown in Table 1. In addition, six patients were reviewed in more detail, because of overlap features and difficulty with classifying the connective tissue disorder. Correspondence. Dr. R. L. Dawkins, Department of Clinical Immunology, Royal Perth Hospital, Perth, WA 6000
Disease
No. Cases
*DIE IF
SLEt RA MCTD PSS MG PM
20 12 7 5 3 13
8 1
0
0 0 0
*
SLE = systemic lupus erythematosus; RA = rheumatoid arthritis; MCTD = mixed connective tissue disease; PSS = progressive systemic sclerosis; MG = myasthenia gravia; PM = polymyositis. t Clinically uninvolved skin. Case Histories Case Number I ( K e n ) is representative of the two patients
seemingly with both RA and SLE. The patient is a 43-yearold woman who has a three year history of seropositive nodular erosive RA. One year ago,' fever, splenomegaly, anaemia, polyserositis and an abnormal urinary sediment developed. Investigations confirmed the presence of pleural and pericardial effusions, high titre antinuclear factor and high anti-DNA binding. Renal biopsy showed mild mesangioproliferative glomerulonephritis. Skin biopsy in this woman, as in the other patient with both diseases, was negative. Case 2 ( M i n ) : A 63-year-old man initially presented with bilateral pleural effusions, an abnormal urinary sediment and a strongly positive speckled antinuclear factor. SLE and MCTD were considered. Skin biopsy showed IgM at the dermal/epidermal junction, but the final diagnosis was metastatic adenocarcinoma.
88
LANGLANDS AND DAWKINS TABLE 2 Junctional staining-cases
of uncertain diagnosis
Diagnoses considered?
RA Mac Ken Min Lon Wat Dev
SLE
MCTD
PM
Ca
Junctional Staining
-
-
1
-
*
-
-
-
i
*
-
-
+ +
* +
+ *
-
+
-
-
-
+ -
+ +
t
Diagnoses considered i possible + likely * eventual diagnosis Abbreviations: RA-rheumatoid arthritis; SLE -systemic lupus erythematosus. MCTD -mixed connective tissue disease; PM -polymyositis; CA-carcinoma.
Case 3 f Lon i : A 50-year-old woman presented in January 1976, with a three year history of fever, a scarring skin rash, alopecia, Raynaud's phenomenon and a one year history of polyarthritis. Four months earlier she had had pleuropericardial pain. On examination she was febrile, and had depigmented skin over light exposed areas. There were areas of scarring alopecia in addition to general loss of hair, and an active erythematous rash over the upper anterior chest. She had peripheral polysynovitis, and 8 cm of splenomegaly. Investigations showed a moderate neutropenia, and high titre speckled antinuclear factor. Negative dermal/epidermal junctional fluorescence on both involved and uninvolved skin and no significant abnormality on renal biopsy suggested a diagnosis of MCTD rather than SLE. Subsequently we have demonstrated marked sensitivity of the antinuclear factor to RNAse treatment. Case 4 ( W a f ) :A 54-year-old woman presented with a six year history of seropositive erosive RA, a one year history of fever, proximal muscle weakness and skin rash and a three month history of dyspnoea and cough. Examination revealed a peripheral polysynovitis of rheumatoid type, moderate proximal muscle weakness, characteristic skin changes of dermatomyositis with a heliotrope eyelid rash, knuckle erythema and erythema, and peeling of the hands and fingers, plus widespread pulmonary crepitations. Investigations showed elevation of muscle enzymes; strongly positive rheumatoid factor; negative antinuclear factor; X-rays and pulmonary function tests consistent with fibrosing alveolitis, confirmed on pulmonary biopsy. Muscle biopsy confirmed myositis. Skin biopsy of uninvolved skin showed dermal/ epidermal fluorescence with anti-IgG conjugate. Case 5 ( D e v ) : A 25-year-old woman with cutaneous, laryngeal and gut attacks of angioneurotic oedema since infancy, with a family history of hereditary angioedema. Twelve months previously she developed a florid discoid skin rash over much of her body and face, together with arthralgia, fever and splenomegaly. Her antinuclear factor was negative; anti-DNA was normal; complement values: C3 > 1 ' 8 g/l, C4 < 0.04 g/l, CHSo < 10 units, and absence of C 1 inhibitor. Skin biopsy of involved and uninvolved skin gave positive fluorescence at the dermal/epidermal junction with monospecific anti-IgG and anti-C3 conjugates.
Discussion
Our findings in SLE are in keeping with those of other workers.', 2 , While the numbers are small,
SUPPLEMENT
1,
VOL.
8
the preponderance of positive results on uninvolved skin was in the group regarded as having significant renal disease.* In turn. this group comprised mainly those patients with the highest anti-DNA binding and lower serum complement levels. Involved skin in the SLE groups was positive in all but one case. Our negative results in patients with PM and PSS are consistent with other reports.6% Those in MCTD are in striking contrast to the report of Levitin in 19753, who described a 500,; incidence of positivity in a group of six patients. The negative results in a total of eight patients justify further prospective analysis of this text to assist in the differentiation of MCTD from SLE. Past reports of negative findings in RA5-7, were substantiated in our patients, and are in stark contrast to the 500,; incidence of positivity reported in an abstract by Baldassare et al. in 1976.4 Even in the two patients who may have both diseases, immunofluorescence was negative. To our knowledge, findings in MG have not previously been reported. The positive findings in the patient with metastatic carcinoma initially suspected of having SLE or MCTD has a precedent." This patient's unusual pattern of fluorescence, being exclusively IgM and somewhat more below the dermaliepidermal junction than usual, raises the possibility of some other antigen, perhaps tumour specific, being fixed in the skin in this case. Conclusion
This test appears useful when positive on uninvolved skin to confirm SLE. The positive staining of involved skin in SLE differentiates this from MCTD with skin lesions. References I . BURNHAM, T. K., NEBLEtr. 7 .R and FiNt. G (1963):The application of the fluorescent anthody technique to the investigation of lupus erythematosus and various dermatoses, J. invest. Derm. 41, 451 2 COKMANF, R. H. (1964): Bound globulin in the skin ofpatients with chronic discoid lupus erythematosus and systemic lupus erythematosus, Lnncet 1,
534. 3. LtViIIN, P. M., WEARY,P. E. and GUILIANO, V. J . (1975): The immunofluorescent "band" test in mixed connective tissue discdse, Ann intern. Mrd. 33, 5 3 . 4. BALDASSARE, A,, WElnS, T., TSAI,C , AU'LAIR.R. and ZUCKNFR, I. (1476): Immune complexes in the skin of patients with rheumatoid arthritis, Arlhr. and Rheum. 19, 788 (abstract). 5. GROSSMAN, J., CALLERMANE, M. L. and CONDEMI.J . J. (1974): Skin immunofluorescence studies o n lupus erythematosus and other antinuclearantibody-positive diseases, Ann. intern. M P d 80, 498.
JUNE
1978
89
IMMUNOFLUORESCENCE IN SKIN
References (Continued) 6. BURNHAM, T . K. and FINE,G . (1971): The immunofluorescent ”band” test for lupus erythematosus. 1 I I employing clinically normal skin. Arch. Derm. 103, 24. 7. DEBOER, D. C , MOSKOWITZ, R. W. and BENO MICHEL(1977): Skin basement membrane immunoflucrescence in rheumatoid arthritis. Arthr. and Rheum 20, 653.
8. GILLIAM, I. N.. CHEANM,D. E., HURD, E. R., STASTNY,P. and ZIPF.M . (1974). lmmunoglobulin in clinically uninvolved skin in systemic lupus erythematosus. Association with renal disease, J. d i n . Invesr 53, 1434. 9. TAN,E. M. and KUNKEL, H. G 11966): An irnmunofluorescent study o f the skin lesions in systemic lupus erythematosus, Arthr. and Rheum. 9, 37. 10. PERCY, I. S. and SYYTH, C J. (1969): The immunofluorescent skin test in Fystenuc lupus erythematosus, J Amer. msd. Ass. 208, 485.
Supplement 1, Vol. 8, Aust. N.Z. J. Med. (1978), pp. 89-93
Some Chemical Aspects of Inflammation: A Brief Overview M. W. Whitehouse From the Department of Experimental Pathology. John Curtin School of Medical Research. The Australian National University, Canberra
Preamble As an acute phenomenon, inflammation is usually beneficial-isolating a noxious agent and preparing the affected tissue for restorative anabolism/cellular renewal. A very readable and extensively documented review of acute inflammation has been published recently.’ We shall restrict our attention here to the chemistry of some less benign aspects of inflammation that command attention in the clinic, particularly : (i) pain, associated with both acute and chronic inflammation ; (ii) loss of function with chronic inflammation, due to either tissue destruction or aberrant repair, or even both together ; (iii) the chemical “fingerprints” that record systemic responses to local tissue injury and may, with acumen, be used to monitor a patient’s ongoing disease and therapeutic response(s). No attempt is made here to survey all known mediators of inflammation (see ref. 1): some may have little clinical significance (serotonin?) and others, though recognised, have still to be fully evaluated as pathophores, e.g. some of the many lymphokines. Immunological and cellular aspects of chronic inflammation are generally excluded from this account (vide other contributions herewith). Special emphasis is given to possible actions of anti-arthritic drugs. Correspondence: Dr. M. W. Whitehouse, Department of Experimental Pathology, John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia
Inhibitors
Membrane perturbation
Phospholipase A=-
Bradykinin
Anti-malarials
PAIN
FIGURE 1 , Effects of some anti-arthritic anti-inflammatory (Al) drugs on prostaglandin (PG) synthesis.
The Value of lmmunofluorescence at the Dermal/Epidermal Junction in Connective Tissue Disorders D. R. Langlands and R. L. Dawkins
From the Departments of Rheumatology and Clinical Immunology, Royal Perth Hospital and Queen Elizabeth II Medical Centre, Perth, Western Australia
Since the pioneering work of Burnham and Fine' and Cormane2, immunofluorescent staining of the epidermalidermal junction has been used as a diagnostic aid for systemic lupus erythematosus (SLE). Recent reports, however, have suggested that junctional staining may be found in mixed connective tissue disease (MCTD)3and in rheumatoid arthritis (KA).s The latter finding is in marked contrast to previous studies5,6 , and the subsequent report by DeBoer and associate^.^ Over the last five years, we have had the opportunity to examine skin biopsies from patients with various connective tissue diseases. In this report we have attempted to determine the diagnostic specificity of junctional staining since biopsies have been obtained from patients with such diverse disorders as SLE, MCTD, KA, progressive systemic sclerosis (PSS), polymyositis (PM) and myasthenia gravis (MG).
Results
Results for the well-defined groups are tabulated in Table 1.,It is noteworthy that while eight of the 20 cases of SLE had positive results, these included five of the seven patients with significant renal disease. The single patient with a positive result in the RA group had only very weak fluorescence with the monospecific anti IgG conjugate. It may be relevant that this patient was on penicillamine therapy, and had ANF and other autoantibodies. TABLE 1 Dermallepidermal junctional fluorescence in connective tissue diseases*
Methods and Patients
Skin biopsies were taken for various indications. In SLE and in those patients in which this diagnosis was entertained, uninvolved skin was taken generally from the forearm. In cases with a suspicion of PM, the skin was obtained from the incision at the time of muscle biopsy. In patients with RA, the biopsy was usually taken because of a skin abnormality such as suspicion of a drug eruption or vasculitis. In the case of patients in which PSS was considered, and in many of those with MCTD, skin was often taken from the phalanges. linmunofluorescence
The specimen was embedded in Ames OCT compound, quick frozen in liquid nitrogen and stored at -70 'C until processed. Cryostat sections 6 1-1 in thickness were stained using fluorescein conjugated antisera to IgG, IgM, IgA and C3. Recent biopsies have also been assessed using C4 and Clq conjugates. Clinical Details Sixty patients were selected for retrospective review, so as to include cases with established diagnoses, as shown in Table 1. In addition, six patients were reviewed in more detail, because of overlap features and difficulty with classifying the connective tissue disorder. Correspondence. Dr. R. L. Dawkins, Department of Clinical Immunology, Royal Perth Hospital, Perth, WA 6000
Disease
No. Cases
*DIE IF
SLEt RA MCTD PSS MG PM
20 12 7 5 3 13
8 1
0
0 0 0
*
SLE = systemic lupus erythematosus; RA = rheumatoid arthritis; MCTD = mixed connective tissue disease; PSS = progressive systemic sclerosis; MG = myasthenia gravia; PM = polymyositis. t Clinically uninvolved skin. Case Histories Case Number I ( K e n ) is representative of the two patients
seemingly with both RA and SLE. The patient is a 43-yearold woman who has a three year history of seropositive nodular erosive RA. One year ago,' fever, splenomegaly, anaemia, polyserositis and an abnormal urinary sediment developed. Investigations confirmed the presence of pleural and pericardial effusions, high titre antinuclear factor and high anti-DNA binding. Renal biopsy showed mild mesangioproliferative glomerulonephritis. Skin biopsy in this woman, as in the other patient with both diseases, was negative. Case 2 ( M i n ) : A 63-year-old man initially presented with bilateral pleural effusions, an abnormal urinary sediment and a strongly positive speckled antinuclear factor. SLE and MCTD were considered. Skin biopsy showed IgM at the dermal/epidermal junction, but the final diagnosis was metastatic adenocarcinoma.
88
LANGLANDS AND DAWKINS TABLE 2 Junctional staining-cases
of uncertain diagnosis
Diagnoses considered?
RA Mac Ken Min Lon Wat Dev
SLE
MCTD
PM
Ca
Junctional Staining
-
-
1
-
*
-
-
-
i
*
-
-
+ +
* +
+ *
-
+
-
-
-
+ -
+ +
t
Diagnoses considered i possible + likely * eventual diagnosis Abbreviations: RA-rheumatoid arthritis; SLE -systemic lupus erythematosus. MCTD -mixed connective tissue disease; PM -polymyositis; CA-carcinoma.
Case 3 f Lon i : A 50-year-old woman presented in January 1976, with a three year history of fever, a scarring skin rash, alopecia, Raynaud's phenomenon and a one year history of polyarthritis. Four months earlier she had had pleuropericardial pain. On examination she was febrile, and had depigmented skin over light exposed areas. There were areas of scarring alopecia in addition to general loss of hair, and an active erythematous rash over the upper anterior chest. She had peripheral polysynovitis, and 8 cm of splenomegaly. Investigations showed a moderate neutropenia, and high titre speckled antinuclear factor. Negative dermal/epidermal junctional fluorescence on both involved and uninvolved skin and no significant abnormality on renal biopsy suggested a diagnosis of MCTD rather than SLE. Subsequently we have demonstrated marked sensitivity of the antinuclear factor to RNAse treatment. Case 4 ( W a f ) :A 54-year-old woman presented with a six year history of seropositive erosive RA, a one year history of fever, proximal muscle weakness and skin rash and a three month history of dyspnoea and cough. Examination revealed a peripheral polysynovitis of rheumatoid type, moderate proximal muscle weakness, characteristic skin changes of dermatomyositis with a heliotrope eyelid rash, knuckle erythema and erythema, and peeling of the hands and fingers, plus widespread pulmonary crepitations. Investigations showed elevation of muscle enzymes; strongly positive rheumatoid factor; negative antinuclear factor; X-rays and pulmonary function tests consistent with fibrosing alveolitis, confirmed on pulmonary biopsy. Muscle biopsy confirmed myositis. Skin biopsy of uninvolved skin showed dermal/ epidermal fluorescence with anti-IgG conjugate. Case 5 ( D e v ) : A 25-year-old woman with cutaneous, laryngeal and gut attacks of angioneurotic oedema since infancy, with a family history of hereditary angioedema. Twelve months previously she developed a florid discoid skin rash over much of her body and face, together with arthralgia, fever and splenomegaly. Her antinuclear factor was negative; anti-DNA was normal; complement values: C3 > 1 ' 8 g/l, C4 < 0.04 g/l, CHSo < 10 units, and absence of C 1 inhibitor. Skin biopsy of involved and uninvolved skin gave positive fluorescence at the dermal/epidermal junction with monospecific anti-IgG and anti-C3 conjugates.
Discussion
Our findings in SLE are in keeping with those of other workers.', 2 , While the numbers are small,
SUPPLEMENT
1,
VOL.
8
the preponderance of positive results on uninvolved skin was in the group regarded as having significant renal disease.* In turn. this group comprised mainly those patients with the highest anti-DNA binding and lower serum complement levels. Involved skin in the SLE groups was positive in all but one case. Our negative results in patients with PM and PSS are consistent with other reports.6% Those in MCTD are in striking contrast to the report of Levitin in 19753, who described a 500,; incidence of positivity in a group of six patients. The negative results in a total of eight patients justify further prospective analysis of this text to assist in the differentiation of MCTD from SLE. Past reports of negative findings in RA5-7, were substantiated in our patients, and are in stark contrast to the 500,; incidence of positivity reported in an abstract by Baldassare et al. in 1976.4 Even in the two patients who may have both diseases, immunofluorescence was negative. To our knowledge, findings in MG have not previously been reported. The positive findings in the patient with metastatic carcinoma initially suspected of having SLE or MCTD has a precedent." This patient's unusual pattern of fluorescence, being exclusively IgM and somewhat more below the dermaliepidermal junction than usual, raises the possibility of some other antigen, perhaps tumour specific, being fixed in the skin in this case. Conclusion
This test appears useful when positive on uninvolved skin to confirm SLE. The positive staining of involved skin in SLE differentiates this from MCTD with skin lesions. References I . BURNHAM, T. K., NEBLEtr. 7 .R and FiNt. G (1963):The application of the fluorescent anthody technique to the investigation of lupus erythematosus and various dermatoses, J. invest. Derm. 41, 451 2 COKMANF, R. H. (1964): Bound globulin in the skin ofpatients with chronic discoid lupus erythematosus and systemic lupus erythematosus, Lnncet 1,
534. 3. LtViIIN, P. M., WEARY,P. E. and GUILIANO, V. J . (1975): The immunofluorescent "band" test in mixed connective tissue discdse, Ann intern. Mrd. 33, 5 3 . 4. BALDASSARE, A,, WElnS, T., TSAI,C , AU'LAIR.R. and ZUCKNFR, I. (1476): Immune complexes in the skin of patients with rheumatoid arthritis, Arlhr. and Rheum. 19, 788 (abstract). 5. GROSSMAN, J., CALLERMANE, M. L. and CONDEMI.J . J. (1974): Skin immunofluorescence studies o n lupus erythematosus and other antinuclearantibody-positive diseases, Ann. intern. M P d 80, 498.
JUNE
1978
89
IMMUNOFLUORESCENCE IN SKIN
References (Continued) 6. BURNHAM, T . K. and FINE,G . (1971): The immunofluorescent ”band” test for lupus erythematosus. 1 I I employing clinically normal skin. Arch. Derm. 103, 24. 7. DEBOER, D. C , MOSKOWITZ, R. W. and BENO MICHEL(1977): Skin basement membrane immunoflucrescence in rheumatoid arthritis. Arthr. and Rheum 20, 653.
8. GILLIAM, I. N.. CHEANM,D. E., HURD, E. R., STASTNY,P. and ZIPF.M . (1974). lmmunoglobulin in clinically uninvolved skin in systemic lupus erythematosus. Association with renal disease, J. d i n . Invesr 53, 1434. 9. TAN,E. M. and KUNKEL, H. G 11966): An irnmunofluorescent study o f the skin lesions in systemic lupus erythematosus, Arthr. and Rheum. 9, 37. 10. PERCY, I. S. and SYYTH, C J. (1969): The immunofluorescent skin test in Fystenuc lupus erythematosus, J Amer. msd. Ass. 208, 485.
Supplement 1, Vol. 8, Aust. N.Z. J. Med. (1978), pp. 89-93
Some Chemical Aspects of Inflammation: A Brief Overview M. W. Whitehouse From the Department of Experimental Pathology. John Curtin School of Medical Research. The Australian National University, Canberra
Preamble As an acute phenomenon, inflammation is usually beneficial-isolating a noxious agent and preparing the affected tissue for restorative anabolism/cellular renewal. A very readable and extensively documented review of acute inflammation has been published recently.’ We shall restrict our attention here to the chemistry of some less benign aspects of inflammation that command attention in the clinic, particularly : (i) pain, associated with both acute and chronic inflammation ; (ii) loss of function with chronic inflammation, due to either tissue destruction or aberrant repair, or even both together ; (iii) the chemical “fingerprints” that record systemic responses to local tissue injury and may, with acumen, be used to monitor a patient’s ongoing disease and therapeutic response(s). No attempt is made here to survey all known mediators of inflammation (see ref. 1): some may have little clinical significance (serotonin?) and others, though recognised, have still to be fully evaluated as pathophores, e.g. some of the many lymphokines. Immunological and cellular aspects of chronic inflammation are generally excluded from this account (vide other contributions herewith). Special emphasis is given to possible actions of anti-arthritic drugs. Correspondence: Dr. M. W. Whitehouse, Department of Experimental Pathology, John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra City, ACT 2601, Australia
Inhibitors
Membrane perturbation
Phospholipase A=-
Bradykinin
Anti-malarials
PAIN
FIGURE 1 , Effects of some anti-arthritic anti-inflammatory (Al) drugs on prostaglandin (PG) synthesis.
