Serum amyloid A regulates granulomatous inflammation in sarcoidosis through Toll-like receptor-2

Abstract

Rationale: The critical innate immune mechanisms that regulate granulomatous inflammation in sarcoidosis are unknown. Because the granuloma-inducing component of sarcoidosis tissues has physicochemical properties similar to those of amyloid fibrils, we hypothesized that host proteins capable of forming poorly soluble aggregates or amyloid regulate inflammation in sarcoidosis.

Objectives: To determine the role of the amyloid precursor protein, serum amyloid A, as an innate regulator of granulomatous inflammation in sarcoidosis.

Methods: Serum amyloid A expression was determined by immunohistochemistry in sarcoidosis and control tissues and by ELISA. The effect of serum amyloid A on nuclear factor (NF)-kappaB induction, cytokine expression, and Toll-like receptor-2 stimulation was determined with transformed human cell lines and bronchoalveolar lavage cells from patients with sarcoidosis. The effects of serum amyloid A on regulating helper T cell type 1 (Th1) granulomatous inflammation were determined in experimental models of sarcoidosis, using Mycobacterium tuberculosis catalase-peroxidase.

Measurements and main results: We found that the intensity of expression and distribution of serum amyloid A within sarcoidosis granulomas was unlike that in many other granulomatous diseases. Serum amyloid A localized to macrophages and giant cells within sarcoidosis granulomas but correlated with CD3(+) lymphocytes, linking expression to local Th1 responses. Serum amyloid A activated NF-kappaB in Toll-like receptor-2-expressing human cell lines; regulated experimental Th1-mediated granulomatous inflammation through IFN-gamma, tumor necrosis factor, IL-10, and Toll-like receptor-2; and stimulated production of tumor necrosis factor, IL-10, and IL-18 in lung cells from patients with sarcoidosis, effects inhibited by blocking Toll-like receptor-2.

Conclusions: Serum amyloid A is a constituent and innate regulator of granulomatous inflammation in sarcoidosis through Toll-like receptor-2, providing a mechanism for chronic disease and new therapeutic targets.

Figure 1.

Serum amyloid A (SAA) forms an integral part of the nidus of epithelioid granulomas in sarcoidosis. (A) Representative photomicrographs of lung and lymph node (LN) tissues from patients with pulmonary sarcoidosis stained by immunohistochemistry (IHC) with a monoclonal antibody (mc1; Dako) specific for fibrillar AA amyloid and SAA (SAA/AA) (top row) or a monoclonal antibody (6F/3D; Dako) to β-amyloid (Aβ) (bottom row) and counterstained with hematoxylin. (B) Representative photomicrographs of pleura, skin, brain (dura mater), and liver stained for SAA/AA. (C) Representative photomicrographs of lung sections from patients with sarcoidosis stained with thioflavin-T and counterstained with hematoxylin and then examined by Nomarski differential interference contrast (panels i and iv) and fluorescence (panels ii and v) microscopy; open arrowheads indicate enhanced fluorescence intensity. Corresponding hematoxylin–eosin (H&E) sections stained for SAA/AA by IHC are shown (panels iii and vi). (D) Top: Soluble SAA levels in unconcentrated bronchoalveolar lavage (BAL) fluid from patients with sarcoidosis and healthy control subjects were measured by ELISA. Bottom: SAA levels in BAL fluid from patients with sarcoidosis grouped according to chest X-ray stage. Data are shown as dot plots with medians (horizontal bars), cohort sizes (n), and nonparametric comparison (brackets) as indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 2.

Greater expression of serum amyloid A (SAA) is found in granulomas from sarcoidosis tissues than in other granulomatous diseases. (A) Representative photomicrographs of lung, colon, or lymph node (LN) tissues from patients with Wegener granulomatosis (WG), hypersensitivity pneumonitis, M. tuberculosis (Mtb) infection, M. avium-intracellulare (MAI) infection, Histoplasma infection, Aspergillus infection, chronic beryllium disease (CBD), hyalinized lymph node granulomas, or Crohn's disease stained for SAA/AA by immunohistochemistry. (B) Representative photomicrographs of lung tissue from subjects with histologically normal lung, cryptogenic organizing pneumonia [COP (ILD)], Wegener granulomatosis, or sarcoidosis are shown for SAA/AA staining (left column) with corresponding red overlay (right column); green overlay is shown for staining of nuclei. (C) Quantification of SAA/AA staining in lung and lymph node tissues expressed as pixels of red area (SAA stained) normalized against pixels of green area (nuclear stained) to account for differences in tissue area in the various samples. The interstitial lung disease (ILD) group includes patients with COP, hypersensitivity pneumonitis, usual interstitial pneumonia, or nonspecific interstitial pneumonia. The infectious lung disease group (INF) includes M. tuberculosis, M. avium-intracellulare, Histoplasma, and Aspergillus infections. Additional groups include chronic beryllium disease (CBD) and Crohn's disease (CD). The control lymph node tissue group (ctrl) includes histologically normal or hyperplastic lymph node. Data are shown as quartile box plots with 10th and 90th percentile whiskers. Group sizes (n) and nonparametric comparisons (brackets) are as indicated (*P < 0.0001). IHC = immunohistochemistry; nl = normal; ns = not significant; sarc = sarcoidosis; WG = Wegener granulomatosis.

Figure 3.

Serum amyloid A (SAA) expression in granulomas localizes to macrophages and giant cells but correlates with CD3⁺ lymphocytes. (A) Photomicrographs of lung, lymph node (LN), and brain (dura mater) from patients with sarcoidosis with heterogeneity in the intensity of SAA staining of neighboring granulomas. (B) Representative photomicrographs (n = 15) of lung (top row) and LN (bottom row) from patients with sarcoidosis stained for SAA (left), CD68⁺ macrophages (middle), and CD3⁺ lymphocytes (right). (C) Correlation of SAA staining as quantified by digital capture microscopy normalized against area of nuclei stained with hematoxylin with the presence of CD68⁺ macrophages or CD3⁺ lymphocytes in the granuloma area from consecutive tissue sections. Analysis of 70 high-power fields from 6 tissue samples is shown. Data are shown as dot plots with lines indicating paired values. Nonparametric correlation is expressed as the Spearman coefficient.

Figure 4.

Serum amyloid A (SAA) activates nuclear factor (NF)-κB through Toll-like receptor-2 (TLR2) signaling independent of TLR4. Shown is the effect of SAA on (A) the human macrophage-like THP-1 cell line with an NF-κB reporter plasmid (THP-1 Blue) differentiated overnight with phorbol myristate acetate (PMA, 10 μg/ml), and then incubated with SAA at 10 μg/ml (or with medium alone or palmitoyl-3-cysteine-serine-lysine-4 [Pam3] at 100 ng/ml) in the presence of polymyxin B (PMX, 10 μg/ml) with or without a 40-μg/ml concentration of blocking antibodies to human TLR2 (aT2) or nonspecific isotype control antibody (Ig). LPS (10 ng/ml) was coincubated with or without PMX as indicated. Activation of NF-κB was detected by a colorimetric assay of secreted alkaline phosphatase (SEAP) activity expressed by the NF-κB–inducible expression plasmid; and (B) HEK-293 cells transfected with a human TLR2 expression plasmid (open symbols) or empty control plasmid (solid symbols) transiently transfected with an NF-κB reporter plasmid (pNiFty) and stimulated with medium alone, SAA, TLR2 agonist Pam3 or M. smegmatis lipomannan (LM; 100 ng/ml), TLR4 agonist LPS, or TLR8 agonist ssRNA (single-stranded RNA, 10 μg/ml). Activation of NF-κB was detected by a colorimetric assay of SEAP activity expressed by the NF-κB–inducible expression plasmid. Data are shown as dot plots of independent experiments with group sizes indicated (n). Medians (horizontal bars) were analyzed by paired (staple brackets) or unpaired (H-brackets) nonparametric methods as indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 5.

Serum amyloid A (SAA) regulates Mycobacterium tuberculosis catalase–peroxidase (mKatG) antigen–driven granulomatous lung inflammation in an experimental model of sarcoidosis. After immunization with recombinant mKatG or an unfractionated cell-free lysate of intracellular proteins from M. tuberculosis (WCL), Sepharose beads linked to the same immunizing antigen or sham-reacted unlinked beads were embolized to the lung of animals in experiments modeled on previously published studies (20, 21). (A) Representative low-power photomicrographs (original magnification, ×10; scale bars, 50 μm) of granulomatous inflammation 4 days after exposure to embolized beads. An index of granuloma size was estimated from the mean radii taken at perpendicular axes (arrows). (B) Granuloma size is shown for 4-day and 3-week time points by box plots with 10th and 90th percentile whiskers. (C) Protein immunoblot with the anti-mKatG monoclonal antibody IT-57 indicating the absence or presence of 80-kD mKatG monomer in mKatG-deleted (mKatG_del) M. tuberculosis strain or the same strain complemented with a functional katG gene (mKatG_comp). Shown is the effect of the presence or absence of mKatG in unfractionated cell-free extracts from these Mtb strains linked to Sepharose beads on the intensity of granulomatous inflammation determined by granuloma size. (D) Antigen-specific responses to mKatG from splenocytes of mKatG-sensitized animals determined by flow cytometry (representative dot plot of CD3⁺CD4⁺ lymphocyte proliferation determined by 5,6-carboxyfluorescein diacetate succinimidyl ester [CFSE] dilution after 6 d of incubation; n = 6) and IFN-γ production after a 24-hour incubation in medium alone, control human albumin (10 μg/ml), or mKatG (10 μg/ml). (E) Representative high-power photomicrographs (original magnification, ×40; scale bars, 50 μm) showing the localization of recombinant human SAA to sites of mKatG–bead–induced granuloma formation after 24 hours; shown are SAA levels in lung homogenates at 24 hours from rats receiving mKatG-coated or uncoated beads. (F) Effect of SAA (10 μg/ml) or Toll-like receptor-2 (TLR2) ligand palmitoyl-3-cysteine-serine-lysine-4 (Pam3, 100 ng/ml) on 24-hour splenocyte production of IFN-γ, tumor necrosis factor (TNF), and IL-10; effect of intravenous injection of SAA (1 mg), transthyretin (TTR, 1 mg), or LPS (50 ng) given immediately before mKatG bead administration on granuloma size at 4 days (lower right). (G) Effect of IP injections of SAA (200 μg) or saline given immediately before orotracheal administration of mKatG beads and at 1 week on granuloma size on Day 4 or 2 weeks in mice immunized with mKatG. (H) Effects of blocking antibodies to TLR2 or similar amount of nonspecific isotype control antibodies on SAA-induced rat splenocyte production of IFN-γ, TNF-α, IL-10, or granuloma size at 4 days (lower right). Data are shown as box plots with 10th and 90th percentile whiskers (B, C, F, G, and H), dot plots with medians (D, F, and H), or as bars indicating mean with standard error whiskers (E). Group sizes (n) and paired (staple brackets) or unpaired (H-brackets) nonparametric comparisons are indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 5.

Serum amyloid A (SAA) regulates Mycobacterium tuberculosis catalase–peroxidase (mKatG) antigen–driven granulomatous lung inflammation in an experimental model of sarcoidosis. After immunization with recombinant mKatG or an unfractionated cell-free lysate of intracellular proteins from M. tuberculosis (WCL), Sepharose beads linked to the same immunizing antigen or sham-reacted unlinked beads were embolized to the lung of animals in experiments modeled on previously published studies (20, 21). (A) Representative low-power photomicrographs (original magnification, ×10; scale bars, 50 μm) of granulomatous inflammation 4 days after exposure to embolized beads. An index of granuloma size was estimated from the mean radii taken at perpendicular axes (arrows). (B) Granuloma size is shown for 4-day and 3-week time points by box plots with 10th and 90th percentile whiskers. (C) Protein immunoblot with the anti-mKatG monoclonal antibody IT-57 indicating the absence or presence of 80-kD mKatG monomer in mKatG-deleted (mKatG_del) M. tuberculosis strain or the same strain complemented with a functional katG gene (mKatG_comp). Shown is the effect of the presence or absence of mKatG in unfractionated cell-free extracts from these Mtb strains linked to Sepharose beads on the intensity of granulomatous inflammation determined by granuloma size. (D) Antigen-specific responses to mKatG from splenocytes of mKatG-sensitized animals determined by flow cytometry (representative dot plot of CD3⁺CD4⁺ lymphocyte proliferation determined by 5,6-carboxyfluorescein diacetate succinimidyl ester [CFSE] dilution after 6 d of incubation; n = 6) and IFN-γ production after a 24-hour incubation in medium alone, control human albumin (10 μg/ml), or mKatG (10 μg/ml). (E) Representative high-power photomicrographs (original magnification, ×40; scale bars, 50 μm) showing the localization of recombinant human SAA to sites of mKatG–bead–induced granuloma formation after 24 hours; shown are SAA levels in lung homogenates at 24 hours from rats receiving mKatG-coated or uncoated beads. (F) Effect of SAA (10 μg/ml) or Toll-like receptor-2 (TLR2) ligand palmitoyl-3-cysteine-serine-lysine-4 (Pam3, 100 ng/ml) on 24-hour splenocyte production of IFN-γ, tumor necrosis factor (TNF), and IL-10; effect of intravenous injection of SAA (1 mg), transthyretin (TTR, 1 mg), or LPS (50 ng) given immediately before mKatG bead administration on granuloma size at 4 days (lower right). (G) Effect of IP injections of SAA (200 μg) or saline given immediately before orotracheal administration of mKatG beads and at 1 week on granuloma size on Day 4 or 2 weeks in mice immunized with mKatG. (H) Effects of blocking antibodies to TLR2 or similar amount of nonspecific isotype control antibodies on SAA-induced rat splenocyte production of IFN-γ, TNF-α, IL-10, or granuloma size at 4 days (lower right). Data are shown as box plots with 10th and 90th percentile whiskers (B, C, F, G, and H), dot plots with medians (D, F, and H), or as bars indicating mean with standard error whiskers (E). Group sizes (n) and paired (staple brackets) or unpaired (H-brackets) nonparametric comparisons are indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 5.

Serum amyloid A (SAA) regulates Mycobacterium tuberculosis catalase–peroxidase (mKatG) antigen–driven granulomatous lung inflammation in an experimental model of sarcoidosis. After immunization with recombinant mKatG or an unfractionated cell-free lysate of intracellular proteins from M. tuberculosis (WCL), Sepharose beads linked to the same immunizing antigen or sham-reacted unlinked beads were embolized to the lung of animals in experiments modeled on previously published studies (20, 21). (A) Representative low-power photomicrographs (original magnification, ×10; scale bars, 50 μm) of granulomatous inflammation 4 days after exposure to embolized beads. An index of granuloma size was estimated from the mean radii taken at perpendicular axes (arrows). (B) Granuloma size is shown for 4-day and 3-week time points by box plots with 10th and 90th percentile whiskers. (C) Protein immunoblot with the anti-mKatG monoclonal antibody IT-57 indicating the absence or presence of 80-kD mKatG monomer in mKatG-deleted (mKatG_del) M. tuberculosis strain or the same strain complemented with a functional katG gene (mKatG_comp). Shown is the effect of the presence or absence of mKatG in unfractionated cell-free extracts from these Mtb strains linked to Sepharose beads on the intensity of granulomatous inflammation determined by granuloma size. (D) Antigen-specific responses to mKatG from splenocytes of mKatG-sensitized animals determined by flow cytometry (representative dot plot of CD3⁺CD4⁺ lymphocyte proliferation determined by 5,6-carboxyfluorescein diacetate succinimidyl ester [CFSE] dilution after 6 d of incubation; n = 6) and IFN-γ production after a 24-hour incubation in medium alone, control human albumin (10 μg/ml), or mKatG (10 μg/ml). (E) Representative high-power photomicrographs (original magnification, ×40; scale bars, 50 μm) showing the localization of recombinant human SAA to sites of mKatG–bead–induced granuloma formation after 24 hours; shown are SAA levels in lung homogenates at 24 hours from rats receiving mKatG-coated or uncoated beads. (F) Effect of SAA (10 μg/ml) or Toll-like receptor-2 (TLR2) ligand palmitoyl-3-cysteine-serine-lysine-4 (Pam3, 100 ng/ml) on 24-hour splenocyte production of IFN-γ, tumor necrosis factor (TNF), and IL-10; effect of intravenous injection of SAA (1 mg), transthyretin (TTR, 1 mg), or LPS (50 ng) given immediately before mKatG bead administration on granuloma size at 4 days (lower right). (G) Effect of IP injections of SAA (200 μg) or saline given immediately before orotracheal administration of mKatG beads and at 1 week on granuloma size on Day 4 or 2 weeks in mice immunized with mKatG. (H) Effects of blocking antibodies to TLR2 or similar amount of nonspecific isotype control antibodies on SAA-induced rat splenocyte production of IFN-γ, TNF-α, IL-10, or granuloma size at 4 days (lower right). Data are shown as box plots with 10th and 90th percentile whiskers (B, C, F, G, and H), dot plots with medians (D, F, and H), or as bars indicating mean with standard error whiskers (E). Group sizes (n) and paired (staple brackets) or unpaired (H-brackets) nonparametric comparisons are indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 5.

Serum amyloid A (SAA) regulates Mycobacterium tuberculosis catalase–peroxidase (mKatG) antigen–driven granulomatous lung inflammation in an experimental model of sarcoidosis. After immunization with recombinant mKatG or an unfractionated cell-free lysate of intracellular proteins from M. tuberculosis (WCL), Sepharose beads linked to the same immunizing antigen or sham-reacted unlinked beads were embolized to the lung of animals in experiments modeled on previously published studies (20, 21). (A) Representative low-power photomicrographs (original magnification, ×10; scale bars, 50 μm) of granulomatous inflammation 4 days after exposure to embolized beads. An index of granuloma size was estimated from the mean radii taken at perpendicular axes (arrows). (B) Granuloma size is shown for 4-day and 3-week time points by box plots with 10th and 90th percentile whiskers. (C) Protein immunoblot with the anti-mKatG monoclonal antibody IT-57 indicating the absence or presence of 80-kD mKatG monomer in mKatG-deleted (mKatG_del) M. tuberculosis strain or the same strain complemented with a functional katG gene (mKatG_comp). Shown is the effect of the presence or absence of mKatG in unfractionated cell-free extracts from these Mtb strains linked to Sepharose beads on the intensity of granulomatous inflammation determined by granuloma size. (D) Antigen-specific responses to mKatG from splenocytes of mKatG-sensitized animals determined by flow cytometry (representative dot plot of CD3⁺CD4⁺ lymphocyte proliferation determined by 5,6-carboxyfluorescein diacetate succinimidyl ester [CFSE] dilution after 6 d of incubation; n = 6) and IFN-γ production after a 24-hour incubation in medium alone, control human albumin (10 μg/ml), or mKatG (10 μg/ml). (E) Representative high-power photomicrographs (original magnification, ×40; scale bars, 50 μm) showing the localization of recombinant human SAA to sites of mKatG–bead–induced granuloma formation after 24 hours; shown are SAA levels in lung homogenates at 24 hours from rats receiving mKatG-coated or uncoated beads. (F) Effect of SAA (10 μg/ml) or Toll-like receptor-2 (TLR2) ligand palmitoyl-3-cysteine-serine-lysine-4 (Pam3, 100 ng/ml) on 24-hour splenocyte production of IFN-γ, tumor necrosis factor (TNF), and IL-10; effect of intravenous injection of SAA (1 mg), transthyretin (TTR, 1 mg), or LPS (50 ng) given immediately before mKatG bead administration on granuloma size at 4 days (lower right). (G) Effect of IP injections of SAA (200 μg) or saline given immediately before orotracheal administration of mKatG beads and at 1 week on granuloma size on Day 4 or 2 weeks in mice immunized with mKatG. (H) Effects of blocking antibodies to TLR2 or similar amount of nonspecific isotype control antibodies on SAA-induced rat splenocyte production of IFN-γ, TNF-α, IL-10, or granuloma size at 4 days (lower right). Data are shown as box plots with 10th and 90th percentile whiskers (B, C, F, G, and H), dot plots with medians (D, F, and H), or as bars indicating mean with standard error whiskers (E). Group sizes (n) and paired (staple brackets) or unpaired (H-brackets) nonparametric comparisons are indicated (*P < 0.005; **P < 0.05). ns = not significant.

Figure 6.

Serum amyloid A (SAA) induces cytokine production from bronchoalveolar lavage cells from patients with sarcoidosis through Toll-like receptor-2 (TLR2) stimulation. Effect of SAA on (A) bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis (sarc, open symbols) or control individuals (ctrl, solid symbols) incubated with SAA (10 μg/ml) or LPS (100 ng/ml) in the presence of polymyxin; cell-free culture supernatants were collected at 24 hours and cytokine levels were determined by ELISA. (B) Gating strategy and representative examples (panel i) of TLR2 expression on CD14⁺ BAL cells from a control subject and a patient with sarcoidosis. Overlay plots of TLR2 staining on CD14⁺ BAL cells representing (panel ii) dot-plot overlay of TLR2 expression in a control subject (green) and patient with sarcoidosis (red) (with the frequency of cells with high TLR2 expression as noted), and (panel iii) histogram overlay of fluorescence intensity of TLR2 staining in a control subject (green shaded area) and patient with sarcoidosis (red outline). (C) Relative expression of human TLR2 on CD14⁺ BAL cells from control subjects and patients with sarcoidosis expressed as mean fluorescence intensity (MFI). (D) Ex vivo expression of cytokines (tumor necrosis factor [TNF], IL-18, IL-10) in sarcoidosis BAL cells stimulated with SAA in the presence of polymyxin (PMX) and blocking antibodies to TLR2 (open symbols) or nonspecific isotype control antibody (solid symbols). Data are shown as dot plots with medians (horizontal bars) and group sizes (n) as indicated, from a total of 34 patients with sarcoidosis and 16 control individuals. Paired (staple brackets) and unpaired (H-brackets) nonparametric comparisons are as indicated (*P < 0.005; **P < 0.05).

Figure 7.

Serum amyloid A (SAA) is a pathobiologic bridge between mycobacterial infection and chronic granulomatous inflammation in sarcoidosis. In this model, SAA is locally induced within antigen-presenting cells (APCs, including macrophages, dendritic cells), within a sarcoidosis granuloma by mycobacterial organisms, and up-regulated in a systemic acute-phase response (1) that is part of an effective killing response that leaves remnants of mycobacterial protein antigens such as mKatG (2). SAA serves as a “seed” for further amyloid-like, noncongophilic protein aggregation and binds to other matrix protein partners to form a nidus for epithelioid granuloma formation (3). SAA and its matrix partners function as a trap for microbial or autoantigens within granulomas while soluble SAA, released from tissue granulomas, serves as a ligand for TLR2 and other innate receptors to regulate Th1-driven epithelioid granulomatous inflammation in part through TNF, Th1 promoting IL-18 and IL-10 (4). Granulomatous inflammation resolves only after concomitant clearance of SAA and local pathogenic antigens (5). In unremitting sarcoidosis, ineffective degradation and clearance of SAA and pathogenic antigens by the polarized Th1 responses leads to chronic inflammation and fibrosis (6).