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The Role of Angiotensin Converting Enzyme (ACE) in Sarcoidosis
|A Clinical and Laboratory Study|
|Dr. Roger K. A. Allen|
|University of Melbourne 1987|
Angiotensin converting enzyme (ACE) is a dipeptidyl carboxypeptidase found on vascular endothelium and other tissues and is produced by epithelioid cells of sarcoid granulomata.
ACE was studied in serum and bronchoalveolar lavage fluid of patients with sarcoidosis and in sarcoid tissue.
Serum ACE was elevated in most (73%) patients with active sarcoidosis, particularly those with pulmonary parenchymal involvement (80%). Serum ACE fell to normal within a fortnight in patients taking corticosteroids and was normal in patients with other pulmonary and granulomatous diseases. All sarcoid patients with hypercalcaemia had elevated serum ACE but normal levels were observed in patients with hypercalcaemia of other causes.
ACE was significantly higher in bronchoalveolar lavage fluid (BAL) of non-smokers with sarcoidosis compared with healthy non-smokers. Lavage ACE but not serum ACE was higher in patients with positive intrathoracic Gallium-67 uptake. Serial studies showed lavage ACE, albumin, IgG and lymphocytes parallelled changes in pulmonary Gallium-67 uptake and lung function.
Three methods of localising ACE in tissues were evaluated. A histochemical technique using fluorescein isothiocyanate conjugates of a synthetic ACE inhibitor, lisinopril (MK521) was not useful because of low affinity. Monoclonal antibodies to human pulmonary ACE produced intense endothelial fluorescence in pulmonary and intercostal arteries. Autoradiographic localisation of human ACE by a synthetic ACE inhibitor, 125 1351A, showed significantly increased ACE activity in epithelioid cells of granulomata of sarcoid lymph nodes. Fibrosis of the granulomata was associated with lower ACE activity in both lymph nodes and serum.
(i) A HISTORY OF SARCOIDOSIS THE DESCRIPTIVE YEARS (1877-1935)
1877 saw the first clinical paper on what was probably sarcoidosis by Jonathon Hutchison, a London Physician. The patient was suffering from gout and "a number of peculiar patches of dark purplish colour on his extremities". The case was published by Hutchison under the title "Case of livid papillary psoriasis". The patient had died in 1875 aged 64 years from kidney disease which in retrospect may have been nephrolithiasis, a known complication of sarcoidosis. Besnier, a French Physician, described in 1889 a condition he named "lupus pernio" which was a violaceous swelling of the nose, ears and fingers but which he considered a different condition from Hutchison's case report. The histology of lupus pernio was described by Tenneson in 1892 as a "predominance of epithelioid cells and a variety of giant cells". One patient, a Mrs. Mortimer, was immortalised by Hutchison who in 1898 described her skin condition as "characterised by the formation of multiple raised, dusty red patches which have no tendency to inflame or ulcerate". He named it "Mortimer's Malady" but did not identify his report of 1877 as being the same condition. Unfortunately he did not obtain a skin biopsy. He believed it to belong to the lupus family, probably of tuberculous origin and accordingly named it "lupus vulgaris multiplex non-ulcerans and non-serpiginosus".
Caesar Boeck, a Norwegian Physician, in 1897 presented a case of a 34 year old policeman with "multiple benign sarcoid of the skin" to the Medical Society of Christiania (Oslo). Boeck recognised the similarities between his case and that of Mrs. Mortimer and described the histological features of the skin biopsy which was characterized by foci of epithelioid cells and giant cells. He believed the disease was a new growth which he described as "perivascular sarcomatoid tissues built up by excessively rapid proliferation of epithelioid connective tissue cells in the perivascular lymph spaces". Over twenty years after Hutchison's initial case report Caesar Boeck published 24 cases of "benign miliary lupoids" which involved a wide variety of organs including lungs, lymph nodes, spleen and bone and was the first person to recognise the systemic nature of the skin disorder described by Hutchison.
Kreibich (1904) first described the connection between bone cysts and lupus pernio, later called "osteitis tuberculosa multiplex cystica" by Jungling (1920). A growing number of clinical descriptions followed in the early part of the 20th century with Darier and Roussey (1906) describing the subcutaneous nodules, Schumaker (1909) and Bering (1910) the iritis, Kutznitsky and Bittorf (1915) describing the clinical and radiological features of pulmonary sarcoid. In 1936 Jorgen Schaumann of Stockholm was responsible for an essay on the systemic nature of the disease and named the condition "lymphogranulomatosis benigna" considering it to have a predilection for the haemopoetic and lymphatic systems and being probably of a tuberculous nature. As early as 1914 he had described the histological pattern as a tuberculoid granulomatous process, presenting minimal phenomena of exudative type and exclusively proliferative character. He believed that "lupus pernio" described by Besnier and Boeck's "sarcoid" were the same condition.
Of all the early physicians who described what we now know as sarcoid only Boeck, and Schaumann recognised the truly systemic nature of the disease. Although Heerfordt, an ophthalmologist of Copenhagen (1909) described uveoparotid fever "febris uveoparotidea subchronica" (Heerfordt's syndrome) which he believed was due to mumps, it was not until the 1930's that physicians such as Bruins-Slot (1936), Longhope and Pierson (1937), Waldenstrom (1937), recognised this syndrome as being due to sarcoid. The last famous syndrome of sarcoidosis to be described this century (Lofgren's syndrome) was by the Swedish physician, Sven Lofgren, who described erythema nodosum and bilateral hilar lymphadenopahy detected on chest radiograph (Lofgren 1946).
It is noteworthy that sarcoidosis was not mentioned in Osler's textbook of medicine even as late as the 10th edition in 1925, six years after Osler's death and nearly a decade after Schaumann's first description of the systemic nature of the disease (Salkin, 1985).
From Boeck's fallacious idea that this disease represented a connective tissue neoplasm or sarcoma we inherit the nonsensical term "sarcoid" (Greek: sarx = flesh). However despite the passage of nearly a century since this ironically fitting misnomer, the true nature and aetiology of the disease remains as elusive as the name is vague.
1935 - 1975
Abnormalities of calcium metabolism were recognised in the late 1930's by Harrel et al (1939) in America and Schupback et al (1941) and Van Crevalt (1941) in Switzerland . The era of clinical description was giving way to a growing understanding of the metabolic and immunologic aspects of the disease. Salversen in Oslo in 1935 described the hyperglobulinaemia and the same year Williams and Nickelson from Boston devised a crude cutaneous test which was the forerunner of the Kveim Siltzbach test. In 1941 Kveim, an Oslo dermatologist, modified this test and observed that the cutaneous papules consisted of epithelioid granulomata, histologically identical to sarcoid tissue. This test was further modified and standardized by Siltzbach, a New York physician, who derived a reagent from a sarcoid spleen from a woman in his city (Siltzbach 1961). Extensive testing of this reagent world-wide in 37 countries between 1960 to 1966 by Siltzbach et al (1967) demonstrated that the disease found world-wide reacted with the same histological features and proved that, what became known as the Kveim-Siltzbach or Siltzbach-Kveim (SK) test, was reliable and of high sensitivity and specificity.
In 1961 a Kveim-Siltzbach reagent was produced in Melbourne , Australia by Hurley and Bartholomeusz from two sarcoid spleens obtained in Victoria . This reagent was also tested world-wide and compared favourably with the reagent of Siltzbach and also a British product. However later studies showed some false positives with a range of conditions such as Crohn's disease, chronic lymphatic leukaemia and other cases of lymphadenopathy (Editorial MJA 1972). The test fell from favour in the 1980's as serum angiotensin converting enzyme became more widely accepted and as transbronchial biopsies via fibroptic bronchoscopy became an integral part of the investigative approach.
Although anergy to tuberculin in sarcoidosis had been described in the early part of the century by Boeck and Kreibich, it was Friou (1952) who rediscovered this and found that the anergy occurred with the range of antigens such as mumps, Candida albicans and trichophyton. The mechanism behind this remained elusive until the 1980's.
The past three decades have seen a growing interest in this disease world-wide. In 1958 in the first World Conference on Sarcoidosis was held in London, U.K. with regular world conferences being held every three years since, the last being in 1984 in Baltimore, U.S.A. A Bibliography on sarcoidosis from 1879 to 1963 containing nearly 4,000 references was compiled in 1964 (Mandel et al. 1964) under the direction of Martin H. Cummings, the Director of the Natural Library of Medicine, U.S.A., who later expanded the service into the medical computerized information system, MEDLARS.
The use of corticosteroids in the treatment of sarcoidosis was introduced in the mid 1950's (Gleckler 1956; McSwiney & Mills, 1956) and its application rationalized by Siltzbach (1958).
Since the early histological descriptions of Schaumann and Boeck there have been only minor additions made to the light-microscopic features of the granuloma. Although the disease cannot be diagnosed on the grounds of histological findings alone, there are many distinguishing features which separate this disease from other granulomatous conditions. Unfortunately there are many conditions whose histology remains non-specific and only adjunctive evidence with which to support a clinical diagnosis. It was in Washington in 1960 that the International Conference on Sarcoidosis rejected a definition of sarcoidosis based on histopathology because "the characteristic histological appearance is not pathognomonic".
The characteristic histological features of the sarcoid granuloma are non-caseating epithelioid cells and Langhan's giant cells clustered together with lymphocytes which form a palisade peripherally. The granulomata usually occur in tissue with monotonous uniformity although this uniformity is not necessarily the rule (Drury 1970). Pinner (1938) described the histological picture thus: "The lesions consist of tuberculoid accumulations of epithelioid cells, frequently but not always, surrounded by a thin layer of lymphocytes. Within the epithelioid follicles, occasional giant cells of Langhan's type are seen. Necrosis and caseation are absent, although single epithelioid cells may show some necrobiotic changes; exudative features and polymorphonuclear cells are absent".
The French physician, Pautrier (1940), stressed the importance of this uniformity in his description of the histology "Un premier point doit etre mis en évidence: la constance des lésions".
Caseous necrosis does not occur in the sarcoid granuloma although small eosinophilic areas of granular necrosis not infrequently occurs in the centre of the granuloma (Mitchell, 1977). This may at times prove difficult to distinguish from true caseation (Dunnill, 1982).
As the granuloma grows older it usually becomes fibrosed with hyalinisation occurring peripherally (Teilum, 1964) and a PAS-positive material gradually accumulates in the epithelioid cells (Teilum, 1956). These granulomata are supplied by vessels whose lumina are usually not oblitered (Teilum, 1964). However sarcoid granulomata often have a predilection for perivascular sites and may cause destruction of elastic laminae of contiguous pulmonary arteries (Dunnill, 1982). Rosen (1978) observed granulomatous angiitis in 69% of 128 open lung biopsies. Pulmonary arterial and venous involvement occurs with equal frequency (Dunnill, 1982).
Unfortunately the intracellular inclusion bodies commonly found in sarcoid granulomata also remain non-specific in nature. Schaumann bodies are basophilic conchoidal, laminated calcified spherules made up of calcium/iron impregnated lipomucoglycoproteins sometimes containing refactile crystals in the centre (Jones Williams, 1960). They occur in epithelioid and giant cells, but have been identified also in chronic berylliosis, Crohn's disease, tuberculosis and extrinsic allergic alveolitis (Jones Williams and Williams, 1968). Another inclusion found in giant cells are asteroid bodies so named because of their stellate appearance. They are microtubules and microfilaments consisting of lipoproteins which are also found in a number of other granulomatous disorders (Cain and Kraus, 1980).
Hamazaki-Wesenberg bodies sometimes called "yellow-brown bodies" or "chromogenic bodies" are bacilliform with a strong PAS-positive capsule varying from 1.5 x 1.0 pm to 10.0 x 5.0 pm in size and found in epithelioid cells and macrophages in sarcoidosis (Dunnill, 1982). Although they also found them in a number of other diseases Doyle et al (1973) found these intracellular bodies in 7 out of 47 biopsy-proven cases of sarcoidosis but considered their ultrastructure to be that of giant lysosomes. Contrary to the opinion of Jones Williams and Williams (1968), Moscovic (1978) regarded them as being L-forms of mycobacteria and implicated them in the aetiology of the disease. Their presence only in sarcoid lymph nodes rather than in other tissues and in only a small proportion of sarcoid patients gives little support to this hypothesis.
An obvious but important feature which distinguishes sarcoidosis from tuberculosis and fungal diseases is the histological absence of acid-fast bacilli and fungi and the failure of such micro-organisms to grow when sarcoid tissue is cultured microbiologically. This absence of micro-organisms remains one of the histological hallmarks of the disease. However, despite these various distinguishing features the histology is essentially non- specific in nature. This not infrequently causes difficulties for the clinician particularly in cases where small transbronchial lung biopsies have been obtained and other diagnoses such as extrinsic allergic alveolitis or silicosis are being considered. There is clearly a need for a more specific histological test for sarcoidosis.
(iii) IMMUNOLOGY OF SARCOIDOSIS
With the help of bronchoalveolar lavage, much has been learned of the immunology of this disease particularly in the lung. The genesis of sarcoidosis is probably the activation of alveolar macrophages by some extrinsic antigen which has so far eluded discovery. Monocytes are then attracted to the lung in huge numbers from the blood and also the bone marrow. In the lung they transform into macrophages which become the building blocks of the granulomata, the epithelioid and giant cells (Boros, 1978; Dannenberg, 1975). This large influx of monocytes to the lung is due to the release of large quantities of monocyte chemotactic factor by T-lymphocytes in the lung (Hunninghake et al, 1979). By contrast, the T-lymphoyctes in the circulation produce relatively small amounts of this lymphokine, macrophage. migration inhibitory factor (Boros 1978). Both the pulmonary macrophages and epithelioid cells are in a metabolically active state and produce a number of enzymes including angiotensin-converting enzyme and lysozyme (Gee et al, 1978; Hinman et al, 1979 a & b; Silverstein et al, 1977 a, b c; Allen et al, 1985).
In active sarcoidosis lung T-lymphocytes are also in an activated state (unlike the circulating T-lymphocytes) and some proliferate locally under the influence of interleukin-1, a product of activated lung macrophages (Hunninghake, 1984). This mass influx of helper T-lymphocytes to the lung may explain the relative lymphopenia and anergy observed in active sarcoidosis. As a consequence of their heightened activity, the T-lymphocytes themselves release many cell mediators such as interleukin-2 and gamma interferon which stimulate the expansion and activation of T-lymphocytes (Pinkston et al, 1983; Hunninghake et al, 1983; Robinson et al, 1985).
Robinson et al (1985) have reported a fourfold increase in the production of gamma-interferon by lung helper T-lymphocytes and alveolar macrophages of patients with active compared with inactive sarcoidosis.
Corticosteroid therapy suppresses gamma interferon release by lung mononuclear cells in these patients. Gamma interferon not only inhibits viral growth but also plays a role in enhancing the immune process generally through stimulation of a broad range of mononuclear cell function. Nevertheless it is unlikely that sarcoid is caused by a viral infection as most viral infections cause the release of non-gamma interferons (Epstein, 1979) which have not been detected in increased amounts in patients with sarcoidosis. It is possible that the primary abnormality in sarcoidosis is a loss of regulatory control of the immune response so that an inappropriate response occurs to a common antigenic stimulus. This then may cause the enhanced release of gamma interferon which has also been found to have very similar characteristics to macrophage activating factor (Zlotnick et al, 1983). I believe it more likely that gamma interferon plays no central role in the pathogenesis of the disease and merely reflects the activated state of both lymphocytes and macrophages in much the same way as ACE does for pulmonary macrophages.
Polyclonal B-lymphocytes also are stimulated to proliferate and release immunoglobulins under the influence of T-lymphocytes, accounting for the presence of the increased concentration of polyclonal gammaglobulins found in bronchoalveolar lavage which spill over into the circulation causing hypergammaglobulinaemia (Hunninghake and Crystal, 1981a). It is unlikely that these antibodies are produced in direct response to the original putative antigenic stimulus.
Through the release of lymphokines such as macrophage migration inhibitory factor (Hunninghake and Crystal, 1981b), the large numbers of recruited monocytic cells are maintained in the lungs, thus perpetuating the dynamic process of granuloma formation. The vast majority of lymphocytes in the lung in active sarcoidosis are "helper" T-cells (Hunninghake and Crystal, 1981b) with twice the proportion of "helper" T-cells in the lungs as in the blood. An increased proportion of "suppressor" T-cells is found in patients with relatively inactive disease as these cells have the effect of dampening down the immune response (Hunninghake and Crystal, 1981b). However the reason for the spontaneous resolution of the disease or for the differing degrees of disease activity seen among patients is not known.
The eventual outcome of all this inflammatory activity is the deposition of fibrous tissue in the lung interstitium produced by fibroblasts proliferating in response to fibroblast growth factor and fibronectin released by local activated macrophages (Bitterman and Crystal, 1981; Rennard et al, 1981). Not surprisingly there is some evidence that the patients who are more likely to suffer functional impairment presumably due to the development of fibrosis are those with a high degree of inflammatory activity in the pulmonary interstitium. This is based on the study of Keogh et al, (1983) who found that an assessment of pulmonary activity based on bronchoalveolar lavage findings and Gallium-67 scan of the lung-fields had some prognostic value. High intensity disease was defined as % lavage T-lymphocytes as greater than 28% with an associated increase in pulmonary increase in pulmonary Gallium-67 uptake; low intensity as less than or equal to 28% T-lymphocytes and/or the Gallium index was normal. Eight-seven percent of the patients in the high intensity had a deterioration of at least one parameter of lung function in the ensuing six months contrasted with only 8% of those with low intensity disease.
There were only five patients with high intensity and fourteen with low intensity disease in the study and the mean deterioration in lung function was 4.1±3.5% for vital capacity and 7.8±1.8% for FEV 1 which is within the variability of the tests. It is noteworthy that diffusing capacity did not change significantly for either groups (high intensity +2.2±2.8%, low intensity +2.3±1.8%) again well within the variability of the test .
(iv) SARCOIDOSIS IN AUSTRALIA
The Australian Contribution
The earliest mention of sarcoidosis in the Australian literature was by Lambie (1940) who described a case of "Besnier-Boeck's Disease" in the Medical Journal of Australia. This was followed soon after by a number of case reports by Frank et al, (1942); Hurley and Hughes (1942); Counsell and Hamilton (1945), Ryan and Counsell (1945) and Isbister (1945). In 1950 Robinson and Pound published an extensive report of thirty cases of sarcoidosis with an excellent review of the historical aspects of the disease. Later that year a review of the current diagnostic and therapeutic approach was published by Robinson. It is ironical that he states:
"Treatment is not satisfactory and results are difficult to assess owing to the nature of the disease. The only form of treatment that appears to affect the process as a whole is the administration of calciferol for long periods" (Robinson, 1950).
The doses used varied from 30,000 to 150,000 international units daily and was continued for months.
Not surprisingly toxic symptoms were described "such as thirst, anorexia, vomiting, nausea, fatigue, malaise, headache, diarrhoea or constipation and abdominal pain" (Robinson 1950). These in retrospect are typical of the symptoms of hypercalcaemia which even then was a recognized side-effect (Anning et al, 1948).
Local deep X-ray therapy was used for skin lesions and nasal sinus and laryngeal involvement. It was even used for lymphadenopathy and intrathoracic disease (Frank, 1942). The current thoughts on the aetiology at that time were summarised by Robinson (1950) as falling into three main groups:
"The first is that it is a form of tuberculosis, and the second that it is a disease sui generis and is possibly due to a virus. The third or eclectic view is that this disease is a non-specific response which may be engendered by a variety of stimuli".
The passage of thirty five years has still not resolved this although the first hypothesis has been virtually discounted. Robinson later published five cases of sarcoidosis in 1952. Marshman from Melbourne published a large epidemiological survey of sarcoidosis in Victoria (Marshman, 1964). Hurley and Bartholomeusz (1968) made a major contribution to the diagnosis of sarcoidosis in this country by the production of a Kveim-Siltzbach antigen. Breslin reported a case of generalized sarcoidosis presenting as skin nodules (Breslin, 1972). McTaggart from Tasmania the following year reported a case of cardiac sarcoidosis (McTaggart, 1973). In 1975 Weston et al reported an unusual case of an Australian patient with pulmonary sarcoidosis and diffuse osteosclerosis of much of his skeleton due to sarcoidosis which was unresponsive to corticosteroids.
Controversy occurred in 1970 with the report by Mitchell and Rees that a large proportion of patients with Crohn's disease had false positive results to the Hurley-Bartholomeusz Kveim antigen. Mitchell and Rees (1969) had claimed that a transmissible agent from both human sarcoid and Crohn's tissue could induce giant cell granulomata in the footpad of mice. This was later refuted (Belcher and Reid 1975).
A perceptive comment regarding the possible mechanism of the Kveim reagent appeared in the Editorial of the Med. J. Aust. in 1972. It states:
"Alternatively, the Kveim test may not depend on the presence of an antigen at all, but granuloma formation may result from the local action of some factor which induces stimulated monocytes to form granulomas. This factor may in turn be produced by the epithelioid cells of the granuloma and lead to the stimulation of adjacent mononuclear cells and so perpetuate granuloma formation".
With our present knowledge of lymphokines and other cellular humeral factors this hypothesis takes on a new meaning. Despite the high incidence of other granulomatous diseases, e.g. tuberculosis and leprosy, in Australian aborigines only one case report of sarcoidosis has been reported (Webling 1978).
The advent of the use of serum angiotensin converting enzyme as a diagnostic tool in sarcoidosis saw a number of publications world-wide in the late 1970s and early 1980s. The first Australian publication on the use and standardisation of this test with the establishment of normal ranges was done by myself and co-workers (Allen et al, 1980). Since then there have been further reports by the same author on the clinical application of the angiotensin converting enzyme in bronchoalveolar lavage fluid (Allen et al, 1983) and also in conjunction with gallium scanning (Gill et al 1983; Allen et al, 1984). These are mentioned in greater detail in later chapters including a new method of diagnosing sarcoidosis by both specific radiolabelled ACE inhibitors (Allen et al, 1985) and also polyclonal and monoclonal antibodies. In 1985, we (Allen and Merory) published the first case report of the successful use of pulse methyl prednisolone in severe widespread neurological and pulmonary sarcoidosis in a patient who had failed to respond to high-dose oral prednisolone. Controlled studies of this new treatment are yet to be done.
Because of the manifold clinical presentations of sarcoidosis, accurate epidemiological studies are very difficult to achieve. There have been numerous such studies into this disease throughout the world over the past thirty years all of which have widely differing incidence and prevalence rates depending on such variables as race, geographical location, age and sex. These are well outlined by Teirstein & Lesser (1983) who comment that this plethora of epidemiological information has added nothing of significance to our understanding of the disease.
As far as Australia is concerned, the most notable study was by Marshman (1964). This ranks as a significant international contribution because of its size and duration, spanning the years from 1959 to 1962 and involving 1,571,011 chest radiographs! It is unlikely that such a huge undertaking will ever be done again in this country or even world-wide. It was done during the period when voluntary mass screening for pulmonary tuberculosis by chest radiograph was performed throughout the state of Victoria as well as most Australian states. Over 400,000 people over the age of 13 years had chest radiographs performed in this state annually out of a total population of 3 million. Marshman reported a prevalence of 9.2 per 100,000 (145 cases diagnosed out of the 1,571,011). Although Marshman uses the word prevalence in his paper he does not take pains to distinguish this from incidence nor to define whether the same patients were studied later in the survey. There was no sex difference and the prevalence was higher in those aged between 15-40 years. Other Australian states he reported had a similar prevalence. Unlike the situation at present, the population at that time comprised nearly entirely of people of European extract over 80% of whom were born in Australia , 2/3 of whom were urban dwellers.
This radiographic method of diagnosing sarcoidosis would underestimate the incidence, possibly by a factor of 50%. A mass survey for tuberculosis in Scotland , 1958-1961, involving 1,709,000 people claimed a prevalence of 8.2 per 100,000 ( Douglas 1983). There was a rising prevalence rate during the course of the study as the clinicians' recognition of the radiological features of sarcoidosis improved. A U. S. Naval study by Sartwell and Edwards (1974) of 1,216,425 recruits entering the Navy between 1958- 1969 showed that only half (65/134) of the cases were discovered by chest radiographs. Similarly a Japanese study by Yanagawa et al (1979) of six nation-wide epidemiologic surveys found that half the 5038 cases reported were discovered by this method.
Thus it would be reasonable to assume that the true incidence of sarcoidosis in Victoria and the rest of Australia is in the order of 20 per 100,000 which brings it into prominence as a disease of economic importance in the community particularly as it affects mainly young people in the prime of their working lives. Its low profile in public consciousness belies its real impact on society. This is highlighted by a report by Balfour (1982) who reported an abnormally high incidence of pulmonary lesions resembling sarcoid granulomata histologically in the bodies of air-crew involved in fatal aircraft accidents. He raised the possibility that there may be a casual relationship between sarcoidosis and these accidents possibly through hitherto unrecognised myocardial sarcoidosis. In the course of this thesis over 100 newly diagnosed patients with sarcoidosis were studied nearly all of whom lived in the Melbourne metropolitan area and mostly within an area of the north-eastern side of the city in the "catchment area" of the two teaching hospitals. During the second study outlined in chapter 4 where over 50 patients were referred for intrathoracic sarcoidosis, one or two new cases were being studied each week. This adds further support to the claim that the disease is hardly a rarity in this community and that its incidence is probably far higher than commonly believed. As sarcoidosis is often chronic or at least subacute, lasting for years, the true prevalence can only be guessed at and is possibly in the order of at least 50 per 100,000. This opinion is in agreement with the findings of Romer (1973) who has done an extensive study of the epidemiological surveys of sarcoidosis in Denmark . He found that the annual radiographic incidence in his country of 5 per 100,000 inhabitants was too low. By his own detailed study in a geographically smaller area he found the annual incidence at least 10 per 100,000 or possibly up to 12 per 100,000. The prevalence of sarcoidosis in neighbouring Sweden based on mass chest radiograph survey alone was 55-64 per 100,000 cases (Bauer & Lofgren 1964). Thus the real prevalence in any country is grossly underestimated and what is diagnosed on mass radiographic survey represents only "the tip of the iceberg".
(vi) DIAGNOSTIC TESTS
ACE In Perspective
The diagnosis of sarcoidosis is, despite all the recent advances in investigative techniques, a diagnosis of exclusion combined with a clinical assessment. Even the presence of non- caseating granulomata on biopsy with no evidence of any associated micro-organisms is still only consistent with the diagnosis rather than being pathognomonic of the condition. It is this deficiency in our ability to diagnose the disease that has motivated much of the work of this thesis.
The Kveim Test
In the 1960's the Kveim test was seen as a significant break through in the diagnosis of sarcoidosis. It provided a highly reliable and relatively non-invasive test which obviated the need to obtain biopsy material from more inaccessible areas such as a mediastinum lymph nodes or lung. As mentioned earlier in the chapter there were several Kveim antigens available world wide, the most well known being that of Siltzbach in the U.S.A. and Hurley in Australia made by the Commonwealth Serum Laboratories. There was also another made in the United Kingdom . However, there was some disenchantment with the Kveim test when a number of reports were published of false positives in a variety of other conditions which could have been confused with sarcoidosis clinically, in particular the number of haematological malignancies (Editorial MA 1972). Despite this the Kveim test still remains in clinical use throughout many centres world wide although to a lesser extent that a decade ago (James et al, 1976).
Anergy to a wide variety of antigens such as tuberculin remains the hallmark of active sarcoidosis which has been recognised since the beginning of the century by both Boeck and Kreibich. However this is clinically of little benefit in reaching the diagnosis as it is by no means universal. At best it serves us further adjuctive evidence with which to support the diagnosis and which may help in assessing whether the disease has become dormant.
Chest radiograph has been an invaluable asset in the diagnosis and management of sarcoidosis particularly since World War II. Mass radiographic surveys world wide have unearthed many sub-clinical cases of sarcoidosis that would have gone unrecognized perhaps until a later stage in the disease. However characteristic the radiographic findings (more often than not they are quite non-specific), the diagnosis still remains to be confirmed by other means. Chest radiograph remains a very useful way to follow the disease in the long term and evidence is mounting that this technique is more practical and sensitive (as well as cheaper and a far lower radiation dose) than Gallium-67 scan in the assessment of active pulmonary sarcoidosis. Unfortunately, as was demonstrated very elegantly by Romer et al (1973) in an epidemiologic study of sarcoidosis in Denmark, radiographic abnormalities are by no means the rule in this disease and is therefore useless as a way of following progress in patients with no overt pulmonary involvement, as for example, may occur in patients presenting with sarcoid uveitis. The purists would nevertheless say that these patients should have radiographs done regularly in the unlikely chance that they should insideously develop pulmonary disease.
The classic or radiographic findings of sarcoidosis consist of bilateral hilar lymphadenopathy with or without a reticulonodular pattern in the pulmonary parenchyma (Kirks et al, 1973). However, either a reticular or nodular pattern may also occur as well as large multiple nodules scattered throughout the lung parenchyma which may resemble "cannon ball" lesions seen with metastatic disease (Kirks and Greenspan, 1973). This plethora of radiographic findings have been classified by the International Congress on Sarcoidosis into three categories:
- Stage 0 - no abnormal radiographic findings
Stage I - lymphadenopathy alone Stage IIa - lymphadenopathy with a diffuse pulmonary involvement
- Stage IIb - diffuse pulmonary disease with no lymphadenopathy
- Stage III - pulmonary fibrosis.
The end-stage of sarcoid involvement in the lungs is a development of pulmonary fibrosis by a process which occurs imperceptably until it reaches a stage where there are definite structural abnormalities such as loss of lung volume, retraction, cysts, bullae, cavities, bronchectasis or segmental atelectasis (McCort & Pare, 1954). This inevitably leads to cor pulmonale and death. Examples of X-rays - sarcoidosis patients
Examples of X-rays - sarcoidosis patients
Computerized Axial Tomography of the Thorax (CT Scan)
The role of computerized CT scan or tomography of the thorax in interstitial lung diseases such as sarcoidosis remains to be fully determined. Although it is an unnecessary and costly investigation it does provide greater detail of the extent of pulmonary involvement than chest radiographs. Putman et al (1977) claimed that there is a closer correlation between CT scan findings and pulmonary function than the chest radiograph. This is not altogether unexpected in view of the increased sensitivity of the test.
Examples of CT scans done on patients with sarcoidosis.
Gallium-67 scanning has, over recent years, come into vogue world wide in the investigation and management of patients with sarcoidosis. Gallium-67 is a cyclotron generated radionuclide with a half life (T1/2) of 78 hours. Its naturally occurring stable isotopes, Gallium-69 and Gallium-71, were first discovered in 1875 by Paul-Emile Lecoq de Boisbaudran who isolated the metal from a zinc blende (Gallium, The New Encyclopaedia Britannica, 1979). Gallium-67 is a group IIIb transition metal that resembles ferric ion in many of its physical characteristics except that elemental gallium has a low melting point (29.80°C). Unlike iron, gallium cannot be reduced in vivo and thus is unable to react with protoporphyrin IX to form heme, remains bound to iron transport proteins such as transferrin, lacto-ferrin, ferritin and siderophores (Hoffer, 1980).
As early as 1949 Dudley and Maddox had demonstrated Gallium-72 citrate could become localized in bones. In 1953 Andrews et al. used Gallium-72 citrate therapeutically for patients suffering from bone tumours. They found at autopsy that the radionuclide also accumulated in soft tissues.
Edwards and Hayes (1969) found that gallium localized in a variety of human neoplasms. Lavender et al (1971) and Littenberg et al (1973) demonstrated its accumulation in a range of non-malignant inflammatory disorders including septic lesions. The earliest reports of abnormal accumulation of Gallium-67 citrate in pulmonary sarcoidosis were by Dige- Peterson et al (1972) and McKusick et al (1973).
Line et al (1981) popularised the use of Gallium-67 citrate scanning in sarcoidosis having demonstrated its use in cryptogenic fibrosing alveolitis three years before (Line et al 1978) using a semi-quantitative method of measuring Gallium-67 uptake by the lungs (the "Gallium index") . It was shown that there was a strong correlation between Gallium-67 uptake and the number of T-lymphocytes recovered at bronchoalveolar lavage. Thus it was considered that Gallium-67 uptake reflected the intensity of alveolitis. Keogh et al 1983) reported that those sarcoid patients whose alveolitis showed a BAL % T-lymphocyte count greater than 28% and positive Gallium uptake in the pulmonary parenchyma would suffer a deterioration in one or more parameters of lung function in the ensuing six months. This has not been confirmed and considerable scepticism about the predicted value of Gallium-67 scanning exists at present. However as Gallium accumulates in alveolar macrophages and lymphocytes in sarcoid alveolitis, it is not surprising therefore that we found a close association between the change in Gallium uptake in the lung over a period of time and the changes in bronchoalveolar cell lymphocyte numbers, proteins and also pulmonary function (see Chapter 6). This observation is also in accordance with the theory that part of the reason for increased intrapulmonary Gallium accumulation is the influx of Gallium into the lung caused by increased capillary permeability. However, it has been my own observation in sarcoid patients who have had bronchoalveolar lavage at the time of the Gallium scan that the vast majority of the gamma activity in the lavage fluid is confined to the cell pellet, suggesting active Gallium uptake by these inflammatory cells.
Gallium-67 is administered as a citrate to facilitate solubilization. After intraveous injection approximately 30% is bound to the plasma proteins and also loosely to albumin and other globulins. The soluble remainder diffuses throughout the extra cellular space or is excreted by the kidneys, one third being excreted in the first week with 10% lost through the gastro- intestinal tract (Bekerman & Vyas, 1976). Gallium-67 is concentrated normally in the nasopharynx, liver, bone, spleen, lacrimal and salivary glands and external genitalia. Total body isotopic uptake is estimated usually 24-72 hours after injection. As the normal lung concentration of Gallium-67 is low, quantitative estimation of abnormal Gallium-67 uptake can be achieved with a computerized gamma camera.
There have been several methods devised of quantitative Gallium uptake in the lungs, all attempts to reduce observer error. There have been several methods devised to quantitate Gallium uptake in the lungs, all of which are designed to reduce observer error. Line et al (1981) devised a semi-quantitative method using a "Gallium Index". With a rectilinear scanner, posterior rather than anterior views were performed because of their diminished sensitivity to difference in body habitus and the presence or absence of breast tissue which takes up the isotope. Gallium-67 uptake in the lungs was derived from the product of image area in pixels and image intensity. The latter was graded "0 to 4" with "0" corresponding to body background as observed in the lower abdomen away from any faecal tracer artifact and "4" usually corresponding to hepatic isotopic uptake (Gallium normally concentrates maximally there). The sum of the products of area and intensity in each region of the lung constituted the Gallium index eg. 100% of the lungs involved at intensity 4 would give an index of 400 units, the maximum possible.
In our study a similar method of quantitation was employed by comparing regional lung activity (the central area of the lungs was outlined by a frame of set dimensions on the computer) with the two reference tissues, liver and thigh, the latter having negligible Gallium-67 activity. Further details are available in Chapter 4.
An analysis of the results of Gallium-67 scanning in the first 26 patients suffering from newly diagnosed sarcoidosis outlined in chapter 4 was made early in this study to assess the value of this technique as a diagnostic tool (Gill et al, 1983). There had been no such studies in Australia at the time as the technique had not been widely used clinically for sarcoidosis. Increased Gallium-67 uptake was observed in the following percentage of cases; mediastinum 55%, lungs 41%, orbits 41%, parotids 24% and spleen 21%. In the control group of 28 patients, six were noted to have orbital activity and 92% had unequivocal gut activity in the 24 hour study compared with only 40% in the sarcoid group. Although 18 sarcoid patients had lung parenchymal abnormalities on chest radiograph, only 10/18 had abnormal pulmonary Gallium-uptake. One patient had a normal chest radiography with abnormal activity in the lung parenchyma.
Late phase (46 and 72 hour) scintiphotographs did not offer any advantage over 24 hour views although both were done.
A mild degree of orbital uptake of Gallium-67 is non-specific finding (Larson et al, 1973) and does not necessarily indicate involvement of the eye or lacrimal glands by sarcoidosis. However, Karma et al (1983) reported three patients with biopsy-proven sarcoid involvement of the lacrimal glands and conjunctive, all of whom had markedly increased uptake in these regions. One patient in our study who had bilateral proptosis due to retro-orbital sarcoidosis, confirmed with CT scan of the orbits, had markedly increased periorbital Gallim-67 activity. Thus it would appear that the presence of florid Gallium-68 activity in the orbits should alert the clinician to the possibility of lacrimal or ophthalmic sarcoid.
In cases where the diagnosis of sarcoidosis was still in doubt, the presence of 67Ga uptake in organs commonly affected by sarcoidosis eg. mediastinum, lungs, spleen, parotid glands and to lesser extent the orbits, assisted in increasing diagnostic certainty especially when combined with an elevated serum ACE. Conversely the absence of any abnormal 67Ga uptake in the body combined with a normal serum ACE made the diagnosis of active sarcoidosis very unlikely. 67Ga scanning also provided an indication of the extent of the disease and organs most severely affected.
Ga-67 scanning - parotid uptake
Klech et al (1983) studied 73 sarcoid patients prospectively to examine the relative sensitivities and specificities of 67 Ga scanning, serum ACE, chest radiograph and blood T-lymphocytes assay in the assessment of sarcoid activity. They observed a significant correlation between serum ACE and 67 Ga score and found that 67 Ga scanning was more sensitive in detecting active disease than serum ACE (92% v 70%). However they concluded that chest radiograph and serum ACE provided the most satisfactory combination of tests for the long-term management of the disease. They considered that Ga-67 scanning and bronchoalveolar should be kept in reverse for those few patients with pulmonary parenchymal disease who sometimes present particularly difficult management problems.
The serial Ga-67 scans of the 18 patients of our study showed that the intensity of isotopic activity in the chest closely paralleled other indices of disease activity such as bronchoalveolar lavage (BAL) lympocyte counts, protein concentrations and respiratory function tests (see chapter 4).
Beaumont et al (1982) found a significant positive correlation between the number of sites of abnormal Ga-67 uptake in sarcoid patients and their level of serum ACE. Line et al (1981) reported a significant correlation between the proportion (%) of T-lymphocytes on BAL and pulmonary Ga-67 uptake expressed by "Gallium Index". However this observation is at variance with Beaumont et al (1982), Gupta et al (1979) and Abe et al (1984). In our study we found that the change in Ga-67 uptake in the lungs correlated with changes in other indices of disease activity including % lymphocytes. One possible explanation for these apparently contradictory observations is a difference in methods of quantitating Ga-67 activity.
Although there are a number of advantages to be gained from Gallium-67 scanning, the technique is non-specific and has a limited role as a diagnostic tool. Its prognostic value has not been confirmed and the radiation exposure to the patient severely limits its clinical usefulness. That the technique provides a reflection of disease activity is beyond dispute.
Examples of full body Ga-67 scanning
Transbronchial Lung Biopsy
Transbronchial lung biopsy has transformed the approach to pulmonary diseases since its advent in 1970 (Anderson and Faber, 1978). In sarcoidosis it has supplanted many of the more traditional methods of obtaining histological confirmation, such as mediastinoscopy of Kveim test, because of its high diagnostic yield, low degree of invasiveness and relative ease of performance (it can be done under local anaesthesia on an out-patient basis). According to Knight et al (1979), the diagnostic yield varies from in the order of 90% in cases of pulmonary parenchymal sarcoidosis to 50% in cases with only hilar lymphadenopathy. This is in accordance with our experience where our usual practice of taking about six biopsies nearly always leads to a positive diagnosis in cases with radiographic evidence of pulmonary parenchymal disease.
Bronchoalveolar Lavage (BAL)
The seventeenth century Oxford physiologist, John Mayo, was well aware of what was, until recently, our inability to study non-invasively the inner workings of the lung, when he wrote.
"The lungs are placed in a recess so sacred and hidden that nature would seem to have specially withdrawn this part both from the eyes and from the intellect; for, beyond the wish, it has not been granted to any one to fit a window to the breast and redeem from darkness the profounder secrets of nature (Mayo, 1668)".
No other recent advance in respiratory medicine has caused such a revolution in the understanding of the immunological basis of pulmonary diseases as bronchoalveolar lavage. From its humble beginnings as a technique devices as a research tool for harvesting pulmonary macrophages from rabbits (Myvrik et al, 1961), bronchoalveolar lavage was over a decade later expanded for human use with the advent of the fibroptic bronchoscope (Reynolds and Newball, 1974). It enables the recovery of large number of viable pulmonary leucocytes for study with low patient morbidity.
The technique involves wedging a fibroptic bronchoscope into a sub-segmental bronchus (usually of the right middle or lingular lobe) and injecting aliquots of usually 20-30 ml sterile warm physiologic saline to a total of 100-150 ml with gentle aspiration after each aliquot. Approximately 50% of the sample is recovered with 10 5 cells/ml. In diffuse disease such as sarcoidosis, the cells recovered, irrespective of the lobe lavaged, are usually representative of the disease process involving the whole lung (Weinberger et al, 1978; Hunninghake et al, 1979). This fact is fundamental for its clinical application. As the fluid recovered contains both cells and proteins, quantitation of differential cell counts and cell concentrations as well as a wide variety of proteins provides a reflection of the activity of the disease in the alveolar and interstitial space.
Normal subjects have a predominance of pulmonary macrophages in their lavage (>90%) with the remaining cells comprising lymphocytes (<8%), neutrophils (G%) and eosinophils (<1%), (Hunninghake et al, 1979). Smoking and bronchitis causes a higher total cell count due to an expanded population of activated alveolar macrophages with a slightly higher proportion of neutrophils (Hunninghake et al, 1979; McLennan et al, 1985). Of the lymphocytes, approximately 75% are T-cells and 8% B-cells. Care must be taken to prevent mucosal trauma and bleeding which may cause spurious elevations in protein and cell concentrations.
The characteristic lavage finding in active pulmonary sarcoidosis is an elevation of the proportion of lymphocytes (Weinberger et al, 1978) the majority of which (>90%) are helper T-cells (Crystal et al, 1981a; Hunninghake and Crystal 1981b). Unfortunately an increase in lavage lymphocytes is also found in a number of other conditions such as extrinsic allergic alveolitis, silicosis, tuberculosis, lymphoma (Reynolds et al, 1977; Crystal et al, 1981b) and lymphoid interstitial pneumonia (Yoshizawa et al, 1984). Thus BAL differential cell count provides only a degree of differentiation from conditions such as cryptogenic fibrosin alveolitis, asbestosis, collagen vascular disease and Histiocytosis-X which are characterised by an elevation in the proportion of neutrophis (Crystal et al 1981b). However the diagnostic value of bronchoalveolar lavage has been reduced by reports of increased proportions of lymphocytes as well as neutrophils in some patients with cryptogenic fibrosing alveolitis. It was these patients who responded best to corticosteroid therapy (Rudd et al, 1981).
The analysis of proteins recovered in lavage fluid in normal subjects has revealed the presence of all the proteins found in plasma indicating that the alveolar-capillary membrane is normally permeable although the concentrations of proteins are extremely low. In active sarcoidosis there is a disproportionate increase in immunoglobulins, particularly IgG and IgM, (Allen et al, 1983), due to local production by an expanded population of polyclonal B-lymphocytes (Hunninghake and Crystal, 1981a; Rankin et al, 1983). Elevations in lavage gamma-globulins also occur in allergic extrinsic alveolitis (IgG and IgM) and cryptogenic fibrosing alveolitis (Reynolds et al, 1977). Thus bronchoalveolar lavage is at best an index of disease activity rather than being of diagnostic value.
ACE is also found in bronchoalveolar lavage fluid in abnormally high amounts in active pulmonary sarcoidosis, being actively secreted by alveolar macrophages (Hinman et al, 1979 a & b; Allen et al, 1983). It is also produced in heightened quantities in the lungs of healthy smokers (Allen et al, 1983), which as its specificity and diagnostic usefulness. Detailed knowledge about -ACE levels in BAL fluid in other interstitial lung disease is not yet available, making its diagnostic usefulness uncertain. How ever, from the study outlined in chapter 4, its activity in BAL of sarcoid patients was found to parallel closely other indices of disease activity. Its role as a marker of macrophage activation has yet to be explored in a number of pulmonary diseases such as carcinoma of the lung and a variety of granulomatous disorders.
Respiratory Function Tests
Bruce and Wassenin (1940) were the first to describe abnormalities in pulmonary function in sarcoidosis, demonstrating the reduction in vital capacity and total lung capacity. Since then more sophisticated tests have shown a number of other functional abnormalities such as reduced carbon monoxide diffusion, lung compliance and increased airways resistance (Cotes, 1979; Winterbauer and Hutchinson, 1980). Although these tests are important in the management of patients with sarcoidosis, they are neither of diagnostic nor prognostic value.
A number of studies have failed to demonstrate a predictable relationship between functional abnormalities, histological findings on open lung biopsy and abnormalities on chest radiography (Young et al, 1966; Carrington et al, 1976; Huang et al, 1979; Winterbauer and Hutchinson, 1980). A normal chest radiography may be associated with abnormal lung function at the outset of the disease while up to 35% of patients with florid pulmonary infiltrates on chest radiograph may have no impairment in lung function (Winterbauer and Hutchison, 1980). Although these discrepancies are not fully understood, studies of open lung biopsy specimens which have been graded either by the density of granulomata and alveolar septal thickness (Young et al, 1968) by the numbers and types of cells in the interstitium (Carrington et al, 1976) or by a combination of these as well as the presence of granulomatous angitis and parenchymal fibrosis (Huang et al, 1979) have all shown a number of statistical correlations with a variety of lung function tests. However a considerable overlap existed with some patients who had little parenchymal disease histologically still displaying abnormalities in pulmonary function.
The pulmonary function test which has the strongest correlation with alveolar wall thickness and numbers of inflammatory cells in the interstitium is diffusing capacity (Young et al, 1968; Snider and Doctor, 1964). Both thickening of the alveolar-capillary membrane (Dm) and reduction of pulmonary capillary volume (Vc) have been considered to be responsible for the reduction in diffusing capacity in .pulmonary sarcoidosis (Hamer 1963; Young et al, 1968; Saumon et al, 1976). A detailed morphometric study by Divertie et al (1976), cast doubt on whether thickening of the alveolar-capillary membrane plays much of a role in reducing diffusing capacity in sarcoidosis. They claimed that this was due almost entirely to ventilation-perfusion inhomogeneities combined with a reduction in the size of the functioning pulmonary capillary bed. Regional studies of pulmonary ventilation (V) and perfusion (Q) using the radionuclide, Xenon, have given support to this concept with significant correlations having been observed between diffusing capacity and the extent of V/Q abnormalities (Renzi and Dutton, 1974; Weitzenblum et al, 1977).
Although endobronchial sarcoidosis is frequently observed at bronchoscopy in my own experience, significant airflow obstruction from large airways disease is unusual. Elevations of airways resistance have been reported in a minority of patients often in association with decreased lung compliance (Coates and Comroe, 1951; Snider and Doctor, 1964; Ting and Williams 1965; Miller et al, 1974; Sharma et al, 1966).
Disease of the small airways is far more common in pulmonary sarcoidosis than disease of the large airways although more technically difficult to detect (Levinson et al, 1977; Miller et al, 1974; Powell et al, 1980). Its detection depends on the frequency dependence of dynamic compliance (Woolcock et al, 1969), the single breath nitrogen test (Anthonisen et al, 1969) and density dependence of maximal flow (Dosman et al, 1976; Hutcheon et al, 1974). Levinson et al, (1977) reported abnormalities of small airways function in all 18 sarcoid patients with restrictive lung defects. Indeed a high proportion of patients with stage 1 and 2 disease and normal spirometry may have small airways disease (Radwan et al, 1978). Crawford and Al-Bazzaz (1982) found the single breath nitrogen test provided useful information about the extent of small airways disease in 35 patients with pulmonary sarcoidosis. They found no convincing reduction in expiratory airflow parameters (FEV1, FVC or IMIMFR) until the patients developed pulmonary fibrosis. Closing volume/vital capacity ratio was elevated in 58% of patients with stage 1 disease and 71% with stage 2. Abnormalities of closing volume were more difficult to interpret in patients with stage 3 and 4 disease because of steepness of the alveolar plateau (phase 3). Although corticosteroid therapy has been shown to produce improvements in diffusing capacity and alveolar-arterial oxygen gradient in patients with non-fibrotic pulmonary infiltrates, they have not, rather surprisingly, been demonstrated to improve small airways function (Renzi et al, 1981). Smoking may have a synergistic effect in the development of small airways disease in sarcoidosis (Dutton et al, 1982).
Pulmonary function tests remain an important way of determining the extent of functional disturbance due to sarcoid lung involvement. However, they, like most of the test already mentioned in this chapter, are non-specific in nature and of little diagnostic value.
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