A Hypothesis to Explain the Genesis of Sarcoidosis

Abstract

Sarcoidosis is a disease of immune cell dysfunction. This review serves to amalgamate the information available into a coherent hypothesis. Recent research has shown that sarcoidosis should not be considered an antigenic induced granulomatous disease alone. The contribution of activation of auto immunity also has to be recognised. The triggering antigens have been narrowed mostly to be derived from Mycobacterial tubercular proteins and Propionobacter acnes. It is possible that they may share a common particle that creates a conformational change in the receptors of Th-1 cells that drives the disease until there is switch to autoimmunity and subsequent development of fibrosis. The role of genetic and environmental factors is also reviewed in this context.

1. Introduction

Sarcoidosis has remained an enigmatic disease since its description by Hutchinson, and subsequent reporting of histopathology by Boeck more than a 100 years ago [1]. Extensive research from many countries has produced voluminous information.

We know that sarcoidosis is an immune disorder where epithelioid non-caseating granulomas form early in the disease with later development of fibrosis if the disease persists. There is a predilection for the lungs and mediastinal lymph-nodes but the disorder can involve any organ of the body. We also know that it involves Th1/Th2/Th17 type responses from the CD4+ T-helper cells, which are typically provoked by difficult to degrade antigens. In addition there is dysregulation of suppressor responses [2]. The problem is that no antigen has been consistently found in the sarcoid tissue, although remnant proteins of the Mycobacterium species have been identified in approximately 20–60% of the tissues tested in various studies. Another putative organism is Propionobacter acnes which has also been detected in upto 85% sarcoid tissue. This is also the only organism to have been cultured from sarcoid granulomas [3]. Although many studies have detected this bacteria, others have not, leaving its causative nature in doubt. Many other antigens like fungi, pollens and other organic material have been considered and discarded for the present. A similar disease can be produced on exposure Beryllium, Zirconium, Silica and other inorganic particles [4].

The Kviem test had shown that a transmissible agent or factor is present in the sarcoid tissue, as this can produce granulomas when injected into the subcutaneous tissues of another sarcoid patient. To be noted is that non-sarcoid tissue when injected into the same patient would not produce a similar response [5]. A tuberculin test at 6 weeks may also produce a similar response in a sarcoid patient, but not other injected proteins. The transmissible granulomagenic factor(GF) can be destroyed by heating the sarcoid tissue at high temperature, but not by heating at lower temperatures [6].

It has also been noted that sarcoidosis may develop in donor lung transplant, if the recipient has sarcoidosis [7]. Sarcoidosis has been reported to occur in recipient of stem cell transplant if the donor had sarcoidosis. Donor immune cells have been identified in the granuloma in this situation [8].

Cases of sarcoidosis have been seen more frequently clustered in families, leading to the consideration that genetic and environmental factors are important. The severity of sarcoidosis and organs involved vary in different regions and ethnic groups. These features have been linked to genetic variations especially HLA class II alleles although other genetic loci are also implicated [9].

In areas of high Mycobacterium tuberculosis transmission, it has been reported that some patients of tuberculosis go on to develop sarcoidosis, the reverse has also been reported. This entity has been termed ‘Tuberculo-sarcoid’. Thus there is evidence that some form of altered Mycobacterial protein is a contender for the initiation and genesis of sarcoidosis [10]. It has been reported in bronchoalveolar lavage fluid (BALF) of sarcoidosis patients that T lymphocytes are very reactive to mycobacterial peptides namely 6-kDa, early secreted antigenic protein (ESAT-6) or catalase peroxide (KatG) [11].

The heavy metal Beryllium and other inorganic substances like silica and Zircon can produce a sarcoid like granulomatous disorder. But these are distinct from sarcoidosis as they can be distinguished by laboratory tests [12].

The fact that anti- inflammatory treatment by Gluco-corticoids or cytotoxic drugs like Methotrexate and not antibiotic or anti tubercular treatment cause improvement and even remission in the disease, highlights that the disease is not caused by an actively multiplying intact organism [13].

2. Granuloma formation in sarcoidosis

Granuloma formation is a specific body response where the immune system has to deal with difficult to degrade or non-biodegradable granulomagenic factor (GF). The granulomagen is first taken up as part of an antigen by the receptors of an antigen presenting cell (APC). These are dendritic cells and macrophages. This foreign substance is recognised as non self, and presented by Th-1 cells that display genetically determined HLA class II antigens to macrophages that ingest the particle and try to destroy it by enzymatic action. Being unable to carry out this task they secrete various factors and interleukins that signal between cells calling upon more cells to enter the involved field. Areas that appear like a ball of nucleated cells develop. Some of the macrophages coalesce to form giant cells and accumulate to form the typical granulomatous tissue. The conglomerate of epithelioid and multinucleate giant cells in the center, surrounded by lymphocytes of the CD4+ type and rare CD8+ T cells as well as some B cells in the periphery has given rise to the terminology epithelioid granulomas. In the case of some granulomas there may be associated vascular involvement and tissue necrosis. In other cases as in M. TUB. there may be caseous destruction of the tissue. The sarcoid granulomas are well formed with distinctive giant cells and necrosis is usually absent [14].

3. Initiation of sarcoid granulomas

There is a distinct polymorphism within genes that code for proteins involved in T cell activation, differentiation, proliferation and persistence which include ANXA11 and NOTCH 4. Risk factors have also been associated within the antigen presentation gene locus 6p21.3 that encode for proteins involved in T cell regulation and antigen presentation involving both HLA and butyrophilin like protein BTNL 2 receptors [15].

On the first exposure to antigen there is the development of tissue hypersensitivity which may or may not be clinically obvious. The macrophages and antigen presenting cells (APC) which are constituted mainly by dendritic cells (DC) are primed. Further exposure to the antigen causes the DC to pick up the antigens and migrate to the local lymph nodes (LN). Here the DC initiate specific T cell differentiation and proliferation. The macrophages also contribute to the early recognition of the antigen in the lung and secrete chemokines such as RANTES, MIP-1 alpha and beta. The Macrophages in addition produce pro inflammatory cytokines such as Interleukin-1 (IL-1) and TNF-alpha, The monocytes highly express Toll like receptor (TLR-2) and also produce TNF-alpha, IL-1beta, and IL-6 when stimulated by ligands ESAT-6, KatG, and endogenous Amyloid A. These activities produce a further influx of monocytes, and LN activated lymphocytes leading to CD4+ alveolitis if the portal of entry is into the lung [16].

The APC with the antigen in the receptor site induce persisting stimulation of the immune cells mediated by HLA related proteins leading to continuous expansion of macrophages and lymphocytes and ultimately to granuloma formation.

4. Persistence of granuloma

The role of TNF-alpha is very important in granuloma formation and persistence. The TNF-alpha produces macrophage activation, migration and leucocyte adhesion. The alveolar macrophages when stimulated by TNF-alpha, and IL-1beta produce CCL20 which is a chemokine attracting DC’s, B cells and specific T-cells towards the site of inflammation. TNF-alpha is also needed for leucocyte adhesion giving rise to high density of cells. This adhesion is induced by intercellular adhesion molecule (ICAM-1).

Following adhesion the histiocyte epithelioid cells and monocyte derived DC’s can fuse to form multinucleated giant cells in the presence of TNF-alpha, GM CSF, IL-17A, CCL20 and interferon -gamma. This process is assisted by natural killer cells that produce INF-gamma.

Differentiation of the T cells is dependent on the cytokines associated with the APCs. Th1 and Th17 differentiation is driven in the presence of INF-alpha, IL-2, IL-17. Further Th1 differentiation also depends on IL-12 and IL-18 whereas Th17 is driven by IL-6 and TGF-beta, but the survival and proliferation of Th17 is also dependent on IL-23. It should be noted that IL-17A/IFN-gamma and IL-17A/IL-4 producing cells are seen to be increased in the peripheral blood of active sarcoidosis. Such a picture is also found in auto immune disease. B cells and plasma cells are present surrounding the granuloma and there are increased serum levels of B cell activating factor (BAFF). These features may select auto reactive B cells leading to antibody production. ANA positivity may be seen in 30–60% of sarcoidosis patients.

There is additional dysfunction of the T regulator cell(T-regs). In Sarcoidosis the T-regs fail to inhibit TNF-alpha, INF-gamma, and IL-2 that contribute to granuloma formation. This ability is restored on recovery.

Recovery from the granulomatous disease may take place with or without tissue destruction depending on the granulomagen. Tuberculosis may heal with fibrosis and distortion of local tissue. On the other-hand sarcoidosis may heal spontaneously or have remission and exacerbations or they may be progressive fibrosis. The destruction of tissue in tuberculosis is due to tissue hypersensitivity but in sarcoidosis the persistent granulomas ultimately cause the invasion of fibroblasts and fibrosis [17].

Anti inflammatory treatment in sarcoidosis elicits a variable response that is dependent on host, environmental factors and possibly the extent of immune dysregulation.

The mode of entry of the granulomagen is commonly into the lung parenchyma from where it may or may not spread via lymphatics to other parts of the body, the other commonly involved organ is the skin [18].

5. The Kviem test

Although the Kviem test is no longer in use now, it has generated much speculation for its enigmatic features. Essentially the test entails harvesting sarcoid material from the spleen of a patient. The tissue is then purified by exposure to various chemicals and heating as well as centrifugation and irradiation. Subsequently it is injected into a suspected case of sarcoidosis. A test is considered positive if a papule develops over 6 weeks, which on biopsy shows non-caseating granulomas.

The test is positive however in about 60% cases in early sarcoidosis and in those after 2 years of disease, true positivity declines to about 20–30%. The waning of this response has not been adequately explained.

The Kviem particle which may be causing the granulomatous response has not been detected despite intense investigation including the use of polymerase chain reaction (PCR} and immunohistochemistry (IHC). However what is known is that Kviem reactivity is nullified if the harvested tissue is exposed to strong acids, alkali, high heat and even centrifugation at high RPM.

The Kviem particle does not appear to lose potency even if tissue from the same papule is injected a few more times into the same subject. Suggesting the persistence of a GF.

At the time when intense immune activity is taking place in the sarcoid patient a seemingly paradoxical cutaneous anergy develops. This can be explained by dysregulated suppressor activity on the appropriate reacting cells.

As the disease progresses, the Kviem test becomes negative probably as the there is a switch in host to activity of the immune system towards autoimmunity against autoantigens and producing fibrosis. At this point the GF polarised T cells diminish as does granuloma formation [19].

6. Genetics in sarcoidosis

The role of genetics in sarcoidosis has been extensively studied in various ethnic groups. The role of class I HLA alleles as well as Class II DR alleles, and non HLA genes have been investigated. From our own studies in Indian patients and their comparison with other ethnic groups we can say that within ethnic groups certain genes either appear to be protective or increase the risk but the same cannot necessarily be extended to other groups. Probably there are genes in linkage disequilibrium that are confounding the issue [20]. The role of genetics is further discussed under Lofgrens syndrome.

7. Non infectious risk factors

Several studies have linked sarcoidosis to the exposure of non infectious agents. It is possible that these agents may act either as adjuvants that trigger the disease in a predisposed individual or are directly causative. Sarcoidosis is more commonly seen in spring from which one could point to pollens or other abundant bio-aerosols that may occur at that time. Of note is a sarcoid like disease seen in fire fighters exposed to combustible substances. Additionally there have been reports of association between toner in printer ink and sarcoidosis. Some of those exposed to the dust from collapse of the world trade center also developed sarcoidosis in a significantly large number, but it is not clear if the inciting agent was dust metal particles or gas. The Sarcoid like disease caused by Beryllium and other inert inorganic substances has already been mentioned [21].

Intuitively inert substances may act as adjuvants rather than actual inciting antigens, because a repeated and adequately heavy exposure would be required for the disease to be initiated and maintained. We already have mentioned that P acnes may multiply in macrophages to increase the antigenic load. Similarly a latent M tub. infection may become active and then after an initial phase of multiplication, the bacteria may degenerate for unknown reasons, leaving enough antigen to incite a granulomatosis response [22].

8. Auto immunity and sarcoidosis

The hall mark of sarcoidosis is the non caseating granuloma and the well described Th1 response. In the core of the granuloma are macrophages, giant cells, CD3+/CD4+ T -helper cells, whereas in the outer portion CD+8 T lymphocytes, T-Regs, fibroblasts and B-lymphocytes predominate. The T-helper type 1 produce gamma-interferon that is essential to induce and maintain macrophage activation leading to chronic disease.

The recent discovery of T-helper 17 cells and its highly specialised sub type Th1/Th17 capable of producing Interleukin 17 and Gamma Interferon both in the local sites and peripheral blood thus reducing the T-regs in the granulomas and increasing them in the systemic circulation has improved the understanding of the immune dysregulation. The 17 cells also contribute to fibrosis and may reflect a switch from external antigen stimulation to auto antigens notably Vimentin [23].

Another notable feature is that Macrophages begin to differentiate to M-2 type which contribute to chronicity by their pro fibrotic and anti inflammatory properties [24].

It seems reasonable to propose that progressive sarcoidosis is characterised by continuous immune dysregulation long after the triggering antigens cannot be detected. This could happen if

1. a granulomagenic factor derived from the triggering antigen persist in the receptor pockets of the Th-1 causing a conformational change that drives the response

2. over time the driving T-helper cells become autonomous due to their genotype even as the GF disappears.

3. there is exposure to self antigens as tissue damage progresses with the development of a switch to auto immunity that drives the disease to fibrosis.

9. The Lofgrens and Heerfordt’s syndrome

The Lofgrens syndrome characterised by fever, arthralgia, arthritis, uveitis, erythema nodosum and bilateral hilar lymphadenopathy is considered to be an early form of sarcoidosis. Many of these cases resolve but a substantial number go on to develop sarcoidosis. A similar syndrome may be seen in tuberculosis called Poncet’s disease [25]. Erythema nodosum can also been seen in leprosy, streptococcal disease, rickettsial disease and exposure to some other infectious organisms. Biopsy of the erythema nodosum shows a septal panniculitis common to all with infiltration with macrophages and lymphocytes. There is no granuloma formation, and no organisms seen. Thus these constellation of signs and symptoms should be considered to be the priming of the immune system, with development of tissue hypersensitivity [26]. A similar reaction in Heerfodt’s syndrome causing parotid enlargement and facial paralysis can be also similarly considered, only the portal of entry being different. A significant point to note here is that many patients may be asymptomatic.

The commonest portals of entry are Lungs and skin of the inciting antigens that contain the GF. Once the first experience of the naive immune system is established, the primed immune cells spread via lymphatics to sites that are pre- determined. These sites are not unique to LS and exhibit similar response to other antigens in which DTH develops. A non specific inflammation is set up. This inflammation may subside over time with treatment or spontaneously. This is attributed to an appropriately functioning immune suppressor system [27].

It is seen that the Class II alleles HLA-DRB-1*03 and DQB1*0201 located in the antigen presenting cells are commonly associated with LS and their presence is prognostically favourable for the reaction to subside. These alleles are seen in patients with LS in Scandinavia. On the other hand in Japan the presence of HLA-DRB1*04 is often associated with Uveitis [28].

In our study in Asian Indians we found these alleles to be rare and LS as well as sarcoidosis associated Uveitis to be uncommon. However we often see Uveitis associated with strongly positive tuberculin test that responds well to anti-tubercular treatment. The genetics of this latter manifestation however have not been adequately studied [29].

Alternatively due to dysregulation of suppressor cells, the disease may progress to the next stage which is of granuloma formation. Associated alleles of the class II genes HLA-DRB1*15, HLA-DRB1*07, HLA-DRB1*14 are often present in the genotype in the APC’s that lead to progressive and chronic sarcoidosis. However in African- Americans who are predisposed to a severe type of sarcoidosis the allele HLA- DRB1*1101 is commonly seen. These alleles express their proteins in the pockets that form in the T helper cells and contain receptors to foreign antigens. In our study we found that HLA- DRB1*15 to be associated with a good prognosis in Asian Indians. But as in other studies, HLA-DRB1*14 and HLA-DRB1*07 were linked to progressive disease [30].

10. Discussion

A number of derivations from the above information can be now presented

1. All antigens that produce Sarcoidosis contain a granulomagenic factor (GF), that persists in sarcoid tissue even if the antigen itself disintegrates. The GF remains intact over long periods of time if it remains in the cell wall but may be disintegrate once in the interstitium.

2. The granulomagenic factor incorporates itself in the pocket of the receptor located in the membrane of the Th1 cells and macrophages. This produces dysregulation and autonomy of the Th1 /Th17 cells. These cells then spread to various organs, where they attract macrophages and cause the macrophages to produce cell signalling interleukins, cytokines and other factors that attract more macrophages to ultimately develop the granuloma.

3. If the sarcoid tissue is now injected into another sarcoid patient the granulomagenic factor is released from dying macrophages and T-helper cells. The GF retains the capacity to promote further formation of local granulomas (for some time} and is incorporated into the host receptors of Th cells.

4. This also explains the granulomagenecity of the tuberculin test. But the occurrence of sarcoidosis in donor lung in a transplanted recipient patient of sarcoidosis is due to the dissemination of the autonomous and dysregulated host Th cells into the tissue.

5. The persistence of disease depends on a number of factors including genetic and environmental factors. After the initial exposure to the antigen, a tissue hypersensitivity develops. The antigen load will determine the GF load. The organism may initially multiply within the host tissues and later degenerate releasing the antigen. The interaction between the GF and the macrophages and Th cells will now determine the dissemination of the cells to other tissues and their progressive dysregulation where suppressor cells become unable to dampen the spread and granuloma formation [31].

6. The GF is probably a nano or submicroscopic particle in the macrophages and in the altered pocket of the T cell receptors [32]. The GF is probably common in structure despite being derived from differing inciting antigens.

7. The further course will depend on the load of the GF in tissues, apoptosis of the cells may cause the disease to regress if GF load is low. But if the GF load is high apoptosis may be prevented by the presence of the GF and granulomas then persist.

8. As mentioned above the T helper cells also become autonomous aided by a dysregulation of suppressor cells, and continue to initiate granuloma formation. Even after the initiating GF has disintegrated, M-2 macrophages may target auto antigens leading to regression of granulomas and development of progressive fibrosis.

9. Anti inf lammatory drugs like glucocorticoids may cause lymphocyte apoptosis leading to extrusion of the GF from macrophages and other granuloma forming cells into the interstitium where it may persist for some time. Glucocorticoids also inhibit the uptake of the GF by macrophages. For this reason there may be a relapse if the glucocorticoids are given for too short a time. Glucocorticoids may also restore the function of the suppressor cells, allowing a restoration of the balance between the effector and suppressor cells.

   Thus the inability of the body to switch off the granuloma response, may be reversed by anti- inflammatory action of glucocorticoids.

10. If the disease progresses unchecked there is now a switch to increased M2 macrophage activity that targets autoantigens and promotes fibrosis. At this point the host loses the Kviem reactivity, as the disease becomes more autoimmune and Th cell activity driven by GF declines. In this regard self antigens have been identified to which the immune system becomes reactive, most notably Vimentin. The protein Amyloid A also plays a role.

11. The reason why the genesis of sarcoidosis has been difficult to understand is because of the changes that take place over time. To put it all together

    1. there is the initial phase of hypersensitivity as seen in Lofgrens syndrome, a similar Poncets disease is seen in Tuberculosis. This signifies an initial exposure to the antigen and priming of the immune system.

    2. At this stage many factors begin to play a role. The major components are genetic most notably the Class II DR alleles. Another factor may be the antigen load. There may be a requirement for high level of antigen exposure for the disease to be progressive. P acnes has been seen to proliferate inside macrophages without causing overt disease but at the same time inducing a spread of bacteria and a strong cellular response. Mycobacteria may do the same but both degenerate for unknown reasons leaving their antigens behind.

    3. If the suppressor cells are effective and the antigen exposure below a threshold the disease will subside when in the early stages.

    4. For the disease to progress, adequate amount GF gets incorporated into the receptors of T-helper CD-4 cells, this promotes the granuloma forming cascade. This also causes a proliferation of the Th-1/Th-17 phenotype. At this point the Kviem test would be positive if this tissue is injected into another sarcoid patient because the GF factor can be taken up by the host cells.

    5. Again over a period of time the concerned T cells develop a permanent conformational change that cause two effects: one an unsuppressed proliferation of the polarised Th cells and second: dissemination of these cells to other organs. Notably by this time the original GF has disintegrated and is no longer needed for the disease to continue.

    6. Persistence of the disease now promotes fibrosis. There is once again a change in the immune state this time from granulomatous to an autoimmune pathogenesis. The macrophages shift to the M2 type and the Th2 responses begin to predominate with the production of auto antibodies.

    7. Thus the reason for the difficulty in understanding sarcoidosis has been to classify it in a single slot i.e. a granulomatous disease, where as it should be seen as progressing through tissue hypersensitivity to granuloma initiated by antigen but maintained by a granulomagenic factor, followed by autonomous T helper cells proliferation causing dissemination of granuloma forming cells to other organs and finally a switch to auto-immune disease leading to tissue fibrosis

    8. The granulomagenic particle cannot be detected by immunohistochemistry or PCR because it is a nano particle derived from the antigens to which the host was initially exposed.

11. The hypothesis

The granulomagen probably has a common structural configuration that is found in several antigens including the Mycobacterial cell wall. This GF which is also transmissible probably incorporates itself into the membrane receptors of the Th-1 cells and inside macrophages causing the secretion of cytokine, chemoattractants and interleukins and increasing the local cellularity of immune cells.

Being deeply embedded into the T cell receptors and thus incorporated as a part of the membrane, the GF is no longer extractable or identifiable even by polymerase chain reaction or immunohistochemistry. We postulate that the macrophages of the sarcoidosis patient that are circulating are primed or sensitised to the granulomagen even though not participating in the granuloma formation. There is also inappropriate suppressor cell activity that causes a loss of skin hypersensitivity to common antigens.

The GF, when injected into the tissue of a sarcoid patient, although present in the membrane of donor Th-1 and inside macrophages may be released by destruction of these very macrophages in the host tissue, after which they get incorporated into the host macrophages that are already primed. In this way granulomas begin to be locally produced by exciting the release of interleukins and signalling factors.

We have deliberately avoided the term ‘Antigen’ and replaced it by ‘Granulomagen’, which we postulate is a submicroscopic particle common to all antigens that have the capability to produce granulomas. In Berylliosis the Kviem test was found to be negative so we can say with confidence that even though the granuloma produced by Berylliosis is similar, it is not the same.

The tuberculin test may also produce granulomas at the injection site, at 6 weeks in a patient of sarcoidosis. This points to the granulomagen present in the tuberculin protein.

12. Conclusion

Sarcoidosis should be considered to be a disease of immune cell dysfunction, provoked in a susceptible individual by an antigen that is a part of a microbe capable of multiplication and subsequent degeneration. The granulomagenic factor contained in this antigen is translocated into the receptors of CD4+ Th1 cells where it generates the granulomatous response. Overtime the Th1 cells of this polarity become autonomous, escaping the immune suppression by T regulatory cells. In the final phase the Th1/Th17 cellular response dominates where the disease switches to self antigens and becomes autoimmune leading to fibrosis.