Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis

Abstract

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.

Figure 1

Scavenger receptor A (SR-A) expression in normal lung, tuberculosis (TB) and sarcoidosis tissue. Immunohistochemistry was performed on paraffin-embedded adult TB lung, child TB lymph node and adult sarcoid lung. Sections were stained with anti-SR-A antibody, followed by streptavidin-biotin immunoperoxidase. The immunoreaction product is brown, and sections were counterstained with Meyer's haematoxylin. (a) SR-A⁺ alveolar macrophages (MØs) in normal lung. SR-A⁺ alveolar (b) and epithelioid (c) MØs on the edge of a granuloma, interstitial MØs (d) in TB adult lung and positive epithelioid MØs (f) on the edge of a granuloma in a child TB lymph node. SR-A^– type II pneumocytes (b) in adult TB lung are shown adjacent to positive alveolar MØs and SR-A^– monocytes in adult TB lung (g) Alveolar MØs (h) in adult sarcoidosis lung are SR-A⁺. Epithelioid (i) and interstitial (j) MØs and giant cells (k) from the edge of a granuloma in sarcoidosis lung are SR-A⁺. Sections adjacent to those in (a)–(k) were negative when anti-SR-A was omitted (l) Magnification, ×400 [(a)–(c), (e) –(f), (h)–(l)] and ×1000 [(d), (g)]. AM, alveolar MØ; AS, alveolar space; EN, endothelium; GC, giant cell; MN, monocytes; TIIP, type II pneumocytes.

Figure 2

Expression of carboxypeptidase-related vitellogenin-like macrophage molecule (CPVL) and angiotensin-converting enzyme (ACE) in normal lung, tuberculosis (TB) and sarcoidosis tissue. (a) CPVL⁺ alveolar macrophages (MØs) in normal adult lung. Alveolar MØs (b) and giant cells and epithelioid MØs (c) surrounding a granuloma in adult TB lung were also positive. Neutralizing anti-CPVL with its peptide prevents signal detection (d) ACE⁺ alveolar MØs (e), epithelioid MØs and giant cells (f) in sarcoidosis lung. ACE⁺ epithelioid MØs (g), ACE-positive (+) and -negative (–) alveolar MØs (h) and giant cells (i) in adult TB lung. ACE⁺ epithelial and goblet cells (j) and endothelial cells (k) in child TB lung. Sections adjacent to those in (e)–(k) were negative when anti-ACE was omitted (l) Magnification, ×400 [(a)–(c), (e)–(j), (l)] and ×200 [(d), (k)]. AM, alveolar MØ; AS, alveolar space; EM, epithelioid MØ; EN, endothelium; EP, epithelium; GC, giant cell; Gob, goblet cell; MN, monocytes; TIIP, type II pneumocytes.

Figure 3

Expression of surfactant protein A (SP-A), surfactant protein D (SP-D) and C-terminal fractalkine (C-Fk) in adult tuberculosis (TB) lung. SP-A⁺ alveolar macrophages (MØs) (a) and type II pneumocytes [(a), (c) ] and SP-A^– giant cell (b) in adult TB lung. SP-D⁺ alveolar MØs (d), type II pneumocytes (f) and SP-D^– giant cell (e) in adult TB lung. C-Fk-positive-ciliated epithelium (g) is shown with strong apical and lateral surface staining. Selected C-Fk-positive type II pneumocytes (h), endothelium and an epithelioid MØ (i) in peripheral areas of a granuloma in adult TB lung. Sections adjacent to those in [(a)–(i)] were negative when anti-SP-A (j) or anti-SP-D (k) was omitted and when anti-C-Fk (l) was neutralized using specific peptide. Magnification, ×400 [(a)–(i), (l)] and ×200 [(j)–(k)]. AM, alveolar MØ; AS, alveolar space; EM, epithelioid MØ; EN, endothelium; EP, epithelium; GC, giant cell; MN, monocytes; TIIP, type II pneumocytes.

Figure 4

Immunophenotype of macrophages (MØs) in relation to granulomatous diseases in the lung. (a) Putative lineage relationships based on antigen expression by different MØs. (b) Antigen expression by epithelial cells, endothelium and MØs, indicating marker antigens and possible cellular interactions.