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Granulomatous disease occurs in 8-22% of patients with common variable immunodeficiency (CVID). We examined the clinical and immunologic information of all 37 of 455 (8.1%) CVID subjects with this complication. The median age at diagnosis of CVID was 26 (2 - 59). 14 had granulomas 1 - 18 years before diagnosis of CVID. In 6 detection of granulomas coincided with this diagnosis; for 17, granulomas were documented later. 54% had lung granulomas, 43% in lymph nodes and 32% in liver. 54% of the group had had autoimmune diseases, mostly immune thrombocytopenia and hemolytic anemia. 24% had had a splenectomy. Nineteen (51.3%) required steroid treatment for granulomas; other immune suppressants were used in some. Over 25 years 28.5% died (median age 37.5), but not significantly more when compared to our CVID patients without granulomas (19.8%). Those with lung granulomas had similar mortality to those with granulomas in other tissues.
Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia, defective antibody responses and recurrent infections [1; 2; 3; 4; 5]. Recurrent pulmonary infections especially for patients with delayed diagnosis or inadequate immune globulin replacement [6; 7; 8; 9; 10], can lead to bronchiectasis, respiratory failure and cor pulmonale, some of the most significant causes of both morbidity and mortality in this immune defect [1; 2; 3; 6; 7]. While hallmark of CVID is that of a B cell defect, many patients also have variable T-cell abnormalities including cytokine defects, poor lymphocyte proliferation[1; 11; 12] and dendritic cell defects [13; 14; 15]. Mutations in selected genes are known to lead to the CVID phenotype, including the inducible co stimulator (ICOS) [16; 17; 18; 19] and CD19 [17; 18]. The role of mutations in the TNFRSF13B gene encoding TACI (Transmembrane activator and calcium modulator and cyclophilin ligand interactor) although demonstrated in a significantly increased number (8%) of patients [20; 21] is somewhat less clear as the same mutations can be found in healthy family members [22; 23]. In order to classify CVID subjects, a number of reports have used various immunological characteristics, particularly B cell functions in response to selected activators in vitro. Most recently, enumeration of peripheral blood switched memory B cells has been used as an indictor of immunologic capacity also showing significant clinical relevance [24; 25; 26; 27; 28].
The development of granulomatous disease or autoimmunity in CVID are two of the most problematic of clinical conditions, as some form of immune suppression may be required for adequate treatment. The granulomatous changes, or what is reported to be “sarcoidosis” is not infrequently diagnosed years prior to the recognition of hypogammaglobulinemia and in these cases, greatly delays the recognition of the immune defect and the revision of the diagnosis to CVID with granuloma [29; 30; 31; 32; 33]. Alternatively, those with known CVID may be found to have granulomata when a tissue is biopsied at a later date. Although granulomas can be found in many organ systems, lungs are the most prominent location [31; 33]. In some patients an intense lymphoid infiltration accompanies the granulomata leading what has been termed “granulomatous lymphocytic interstitial lung disease” (GLILD) [33; 34]the presence of which may be associated with a poorer outcome . For unclear reasons, granulomatous disease is often found in associated with autoimmunity, in particular, immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia (AIHA) . These episodes may occur years before the discovery of granulomas in any tissues. Here we outline the clinical and immunological data of 37 CVID patients in whom granulomas were documented in one or more organ systems and compare the mortality of these subjects to other CVID subjects.
The clinical and immunologic information of a total cohort of 455 patients with CVID were surveyed. The diagnostic criteria for CVID included reduced serum IgG, IgA, and/or IgM by two or more confidence intervals below the normal ranges for ages and antibody deficiency, verified by lack of protective titers to vaccine antigens, based on laboratory reference values. [1; 2; 4]. Subjects who had other causes of congenital or acquired hypogammaglobulinemia were excluded. Serum IgG, IgA and IgM levels at the time of diagnosis, before immunoglobulin replacement, were obtained. Of the group, current medical information was available for 370 subjects; this included 76 subjects who had died in the follow-up period of 25 years. For those patients in whom granulomas had been diagnosed, a review of medical records and pathology reports was performed to verify the finding of granulomas and determine the tissues involved. The results of additional tests, such as pulmonary functions and radiological studies, including those obtained in the course of our evaluations, were reviewed and additional tests obtained where required.
T cell proliferative responses to mitogens (phytohemaglutinin, concanavalin and pokeweed mitogen) were determined by standard methods and compared to results for the laboratory controls. For more recently referred subjects, the percentages of T, B cells, total memory B cells (CD27+CD19+) and isotype switched memory B cells (CD27+IgM- IgD- CD19+) were quantitated by flow-cytometry, using freshly isolated peripheral blood mononuclear cells. Subjects with 0.4% or fewer of isotype switched memory B cells, as based on the number of perpheral blood mononuclear cells, were considered to be Group 1 while those with greater than this number as Group 2 subjects, as previously described [24; 26; 27] For comparison, normal controls of age 18 or older (n=35) had levels of isotype switched memory B cells between 18.2% and 31%.
The median levels of serum immunoglobulins were determined. For patients whom the immune globulin levels were defined as less than a given number, the whole number under this level was used, such as 6 mg/dl in place of < 6.7 for calculations. The Mann-Whitney test was done to compare lymphocyte proliferation results between subjects with granuloma and those without. The Chi square test was used to determine differences between the incidence of autoimmunity in CVID subjects with and without known granulomas; this test was also used to determine differences between the mortality of subjects with granulomas as compared to all other CVID subjects not found to have such lesions. The log rank test was performed to determine if patients with known pulmonary granulomas had increased mortality as opposed to those with granulomas elsewhere. (Prism 4; Graph Pad Software).
Of the 455 subjects with CVID, 37 subjects (8.1%) had been found to have granulomas in one or more tissues. This group consisted of 29 (78%) Caucasian, 5 (14%) African American, 2 (5%) Hispanic and 1 (3%) Asian patients. For the group as a whole, 92% are Caucasian, 2.7% African American, 4% Hispanic and 1% are Asian patients. Of these, two had family history of CVID or IgA deficiency. The age of these patients at the time of diagnosis with CVID ranged between 2 to 59 years (median 26); 24 out of 37 (64.8%) were female (Table-1). (For the group of CVID subjects as a whole, 57.5% were female.) Granulomas had been detected in 14 of these patients, 1 to 18 years before the diagnosis of CVID was established. In 6 patients of the group, the detection of granulomas coincided with the diagnosis of the immunodeficiency; in the remaining 17, granulomas were found after the diagnosis of CVID. Granulomas were documented in the lungs in 20 of these subjects (54%) and 12 (60%) also had granulomas in other organs, the liver and the lymph nodes being the most common (Table-1). The other locations in the order of frequency were lymph nodes (16), liver (12), skin (7), spleen (5), bone-marrow (2), brain (2), retina (2), small bowel (1) and kidney (1). On lung biopsy, 6 subjects of the group (cases 5,8,13, 14, 28 and 36) had a dense lymphocytic infiltrate in the lungs and were diagnosed as having lymphocytic interstitial pneumonia. Four patients (cases 1,12,19, 22) also were also found to have a lymphoid infiltration accompanying the granulomas found on liver or spleen biopsies. Subject 29 was found to have granulomas on skin biopsy done after an acute antibiotic-induced drug eruption; he has no other known granulomas. The granulomas in various organs were very large, as for one subject in whom the spleen and surrounding mesenteric tissues formed a granulomatous mass (Figure 1, subject 15) or multiple, as for subject 19 who had numerous granulomas studding the surface and interior of the spleen (Figure 2.) Some granulomata were solitary, as shown for Subject 26 in whom only one granuloma was found in the bone marrow examination done for ITP (Figure 3). An organism was not identified in any case using standard stains and cultures.
Twenty two patients (59%) have had moderate to severe respiratory symptoms with chest radiographic abnormalities including nodular changes, bronchiectasis and/ or diffuse interstitial changes leading to reduced vital capacity and impaired diffusion capacity of carbon monoxide. One of these had a bilateral lung transplantation and 3 others had been considered for this procedure. One of these subjects (#19) has severe hepatopulomary syndrome and hepatic transplantation has been considered. While 7 subjects (19%) had neither clinically significant chronic respiratory symptoms nor radiological or functional evidence of pulmonary disease, the remaining have either localized bronchiectatic changes or other interstitial abnormalities with retained lung functions.
Serum immunoglobulin levels at diagnosis and peripheral lymphocyte phenotypes are shown on Table-2. Median IgG, IgA and IgM levels for the group were 210 mg/dl, 6 mg/dl and 19 mg/dl, respectively. Six of the 29 (20.6%) patients investigated had an inverted CD4+/CD8+ T cell ratio. While lymphocyte proliferative studies also were not performed in each case, 24 of the 33 tested (72.7%) had reduced T cell responses to at least one of these mitogens. (On Table 2, shown as underlined values.) Comparing these lymphocytes responses to other CVID subjects in this group who did not have granulomas, lymphocyte proliferation analyses were not significantly different. For 22 subjects, the percentage of isotype switched memory B cells (CD27+ IgM-IgD-) was obtained; these revealed very low levels (0.4% or fewer) for 20 of these subjects; 9 had switched memory B cell percentages of 0%. Two others of the group had more than 0.4% .
Twenty of these 37 (54 %) patients had one or more episodes of autoimmunity. Ten of these subjects had a history of ITP, 4 had had AIHA, 5 patients had had both ITP and AIHA at different times. (Table-3). Four subjects (Cases 2,10,30,35) had multiple episodes of these autoimmune cytopenias. In contrast, for the 418 other patients, with no known granulomas, one or more autoimmune conditions were known for significantly fewer subjects, 76 subjects (18%.) (p= 0.001) (This includes predominantly ITP and/or AIHA (55 subjects), but also rheumatoid arthritis (8), insulin dependent diabetes (3), alopecia totalis (3), anti-IgA antibodies (3), systemic lupus erythematosus (2), vasculitis (2), juvenile rheumatoid arthritis (1), primary biliary cirrhosis (2), pernicious anemia(2), neutropenia (2), thyroiditis (2) and myasthenia gravis (1). Steroids, either orally or intravenously, were given to 19 of the 37 patients to treat acute autoimmune disease (Table 3.) Splenectomy was performed in 9 (24.3%) of these patients, either for uncontrolled autoimmunity, splenic enlargement (2) , or suspicion of lymphoma (1). One subject received rituximab (Case 2) with resolution of autoimmunity. Nineteen patients (51.3%) had also been given steroids, with or without other therapies, for reducing or controlling granulomas in lungs, nodes, liver, brain or skin. Seven of these subjects were also given one or more additional immune suppressants: hydroxychloroquine (5 subjects), cyclosporine (3 subjects), azathioprine (2 subjects), methotrexate (2 subjects), infliximab (1 subject), and etanercept (1 subject) were also used either alone or in different combinations. None of these additional therapies was of clear benefit in the treatment of granulomatous disease. (Table-3). (In contrast of the 418 subjects with no known granulomas, splenectomy had been performed in 20 subjects, 5% of the group.)
Ten out of the 35 (28.5%) patients with granulomas for whom current information is known, have died, at median age of 37.5 years, over the 25-year follow-up. (Table-4). Seven of these patients died of respiratory failure related to granulomatous lung disease and one died of liver failure stemming from granulomatous liver disease. Of the group who died, LIP had been found in one. (#28) One patient died shortly after lung transplantation due to surgical complications. One patient, not on any immune suppressants, died of progressive multifocal leukoencephalopathy (PML). The differences in mortality between patients with pulmonary granulomas as compared to subjects with granulomas elsewhere, was not significant (p=0.230.) For the total group of 455 subjects, current follow-up was known for 370 subjects. Using this group as the denominator, we did not find a significant difference in mortality for subjects with granulomas as compared to those without, of which 19.8% died in this interval (p=0.68).
Granulomatous disease occurs in between 8 to 22% of subjects with CVID [1; 30; 31; 33; 35] an association still not commonly recognized by physicians [29; 30; 31]. In fact when granulomas are discovered first, the diagnosis of CVID is often delayed as the patient is considered to have uncomplicated sarcoidosis. Tissue biopsy may be required not only for diagnosis of granulomas but also to differentiate these lesions from other complications such as lymphoid hyperplasia or lymphoma, which also occur in CVID [36; 37]. Lungs, lymph nodes and spleen are the most commonly affected sites, although other organs such as skin, liver, bone-marrow, kidney, gastrointestinal tract and brain can be involved [30; 31; 32; 33; 38; 39; 40; 41; 42]. In our cohort, lungs were the primarily affected site, followed by lymph nodes and liver. In six of our subjects, a lymphocytic interstitial infiltrate accompanied the granulomas, a combination that may contribute to respiratory insufficiency and death [33; 34]. A recent study reported a median survival of 13.7 years for CVID patients with granulomatous/lymphoid interstitial infiltrates, as compared to 28.8 years in those who did not have this complication . In our cohort, 10 patients (of the 35 for whom we have current data) died in the follow-up period of 25 years; only one of whom also had LIP. Six of these were complicated by respiratory insufficiency resulting from granulomatous interstitial lung disease although pneumonia was a precipitating cause in five. We could not verify that patients with known granulomas in lungs had a shorter survival than those who had granulomas in other tissues. This difference may be due to the bias introduced by the choice of tissue that was examined; if granulomas were found in an enlarged lymph node or liver, the lungs may not have been biopsied as well, unless a clinical reason required this procedure. In addition, the mortality for these subjects with granulomas was not significantly different from the mortality for the other members of the CVID group in whom granulomas were not found (19.8%.) This is likely to be due to the nature of this cohort of subjects, as it is now clear that patients with non-infectious complications, potentially over-represented in this population, have an increased mortality when compared to subjects who have only recurrent infections .
For unclear reasons, granulomatous changes are associated with autoimmunity in CVID . In this cohort, more than half had been diagnosed with an autoimmune condition, ITP or AIHA being the most common. This is very significantly increased in comparison to the percentage of autoimmunity (18%) known in the remaining CVID population, not specifically noted to have granulomas . CVID subjects with granulomas or autoimmunity are also likely to have a significant deficiency of isotype switched memory B cells [24; 25; 26; 27] suggesting that most profound lack of B cell maturation is likely to be associated with the abnormal genetic, cellular or cytokine environment that supports granulomata formation. T cell defects in CVID have also been associated with the formation of granulomas in tissues, including an expansion of CD8+ T cells and lack of CD4+ T cells with defective T cell proliferative responses [31; 43] as shown for many of the subjects examined here.
In general, granulomas are known to develop in response to microbial or insoluble agents. Since the lungs are commonly involved in CVID, the search for an environmental antigen triggering this reaction is always worthwhile. The granulomas in CVID are usually of non-caseating nature, although caseating granulomas have also been reported [30; 31; 44] raising the possibility of an infectious etiology as the original trigger. A case of CVID with granulomatous and lymphoproliferative disease following an acute Toxoplasma gondii infection has been reported in a 12 year-old girl .The human herpes virus 8 (HHV8) has been observed in pulmonary samples and bone marrow of some CVID patients with granulomatous lymphocytic interstitial lung disease . However, in most patients no microorganism has been identified, as for the subjects reported here, although in time molecular techniques may shed more light on such lesions. In the case of patient 29 here, granulomas were only found on skin biopsy done for an acute antibiotic drug reaction, suggesting that granulomas can be formed in subjects with CVID, in other settings of inflammation.
In sarcoidosis, a model for understanding granuloma formation, the cardinal histologic feature of granuloma is an excess of CD4+ T cells, potentially enriched in oligoclonal alpha beta T cells; these appear to interact with antigen presenting cells to initiate the granulomatous tissue reaction . Activated CD4+ T cells produce both IL-2 and INF-gamma, also leading to TNF-alpha production [47; 48]. Cultured alveolar macrophages release high levels of TNF-alpha in sarcoidosis, which is correlated with disease progression . TNF-alpha has been shown to highly expressed within granulomas in tuberculosis  The crucial role of TNF-alpha in the maintenance of granuloma formation has been demonstrated in several experimental models which indicate that in the absence of TNF or TNF receptors, there is defective granuloma formation, due to inefficient dendritic cell function and/or lack of cellular infiltration essential for containing the infection [51; 52; 53]. The role of IFN-gamma in central necrosis has also been suggested, but its overall role in the development of granulomatous disease is unclear . Cytokine abnormalities have been previously noted in CVID. T cell INF-gamma secretion was upregulated in some patients especially in subjects with reduced numbers of naive CD4+ T cells [43; 54]. While intracellular IL-12 was increased in monocytes in one study of subjects with CVID , IL-12 production by monocytoid DCs was found defective [13; 14]. As TNF-alpha plays an important role in granuloma in model systems and possibly human diseases, this cytokine has attracted the most attention in CVID. From older data, 11 of 24 CVID patients with reduced CD4+ T cell counts, increased neopterin levels and splenomegaly, had significantly elevated serum levels of TNF-alpha and both forms of soluble TNF (p75 and p55) receptors (sTNFRs) . Mullighan reported granulomas in 20 of 90 patients with CVID (22%); 8 of these 20 (40%) had an unusual TNF-alpha allele (TNF +488A)  but increased TNF-alpha production was not actually examined in these patients. Another factor potentially enhancing the formation of granulomas in CVID might result from the defective TLR9 signaling shown to occur in this immune defect  as TLR9 knockout mice have increased granuloma formation on challenge with mycobacterial antigens .
Appropriate treatment of granulomas in CVID presents a particular challenge, as some form of immune suppression may be required, always undesirable in the presence of an existing immune defect. Unfortunately immunoglobulin replacement has shown little effect in controlling granulomatous changes, however, with one exception . When immunosuppressive therapy is required in clinical grounds, steroids have been the first choice, however, additional agents may be needed if the dose of steroid required is more than desirable or the length of treatment is prolonged. Hydroxychloroquine may be an alternative treatment due to inhibitory effect on monocyte-macrophage activation, antigen presentation and TNF-alpha release . Cyclosporine has also been used successfully in some patients for LIP , and was used for two subjects in this series with unclear benefit. TNF-alpha -blocking agents (infliximab and etanercept) have attracted particular attention because of the putative role of TNF-alpha in granulomatous disease in CVID, and beneficial effects noted in some cases of sarcoidosis [61; 62]. These therapies have also been used with some success in case reports of cutaneous or systemic [63; 64] granulomatous diseases in CVID. However, which subjects should be given this therapy, and when, remains unclear; collection of further data on this subject is required. It was given to two of our subjects with no improvement. Mycofenolate mofetil (CellCept) may be an alternative option especially in patients diagnosed with granulomatous and lymphocytic infiltrations as it may exert a preferentially suppressive effect on activated T cells, inhibits expression of adhesion molecules and inhibit nitric oxide (NO) synthesis . However, immunosuppressive therapy may expose patients to a risk of opportunistic infections and possibly malignancy, a risk in CVID [1; 36; 66]. In pulmonary granulomatous lymphocytic lung disease, lung transplantation has been performed (as for case #6 here) however granulomas can recur in the transplanted lung . Although hypercalcemia is common in sarcoidosis presumably due to increased 1,25-dihydroxyvitamin D3 production by activated macrophages , it appears unusual in subjects with CVID with granulomas, with few exceptions  and case #14-here]. In these cases, steroids or hydroxycloroquine can reduce the serum calcium. While the presence of granulomas can promote tissue damage leading to the need for intervention, it should be noted that 7 subjects in this cohort, with granulomas documented in tissues 1 to 18 years previously, have never required any treatment.
In summary, granulomatous disease is a relatively unusual complication of CVID and is still poorly recognized. The causes of this tissue reaction remain unknown, as the cellular and cytokine abnormalities are still not well elucidated. Where required on clinical grounds, the best therapeutic approach is to control the inflammatory cascade by using immunosuppressive and/or antiinflammatory agents as sparingly as possible. Clinical trials to gather sufficient data on subjects with this complication, coupled with additional investigation of the pathogenesis, may help to better guide therapeutic options.
#This work was supported by grants from the National Institutes of Health, AI 101093, AI-467320, AI-48693 and NIAID Contract 03-22 to CCR;