Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue

Abstract

To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll-like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue. We found that TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and dendritic cells. In addition, TLR1 and TLR2 were expressed in mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.

Introduction

The immune response to microbial infection can be segregated based on the speed and duration of the response. The innate immune response is rapid, allowing for early detection of microbial pathogens and control of infection. Cells of the innate immune response, including macrophages, dendritic cells (DCs) and neutrophils, express pattern-recognition receptors that recognize and respond to microbial pathogen-associated molecular patterns such as lipopolysaccharide (LPS), lipoproteins, and bacterial DNA. Pattern-recognition receptors are non-clonal, i.e. present on all cells of a class, and do not depend on immunological memory because they are germline encoded. In contrast, the adaptive immune response is delayed in onset of activation and involves immunological memory. The adaptive immune response contains receptors that are highly specific, require germline rearrangement (e.g. T- and B-cell receptors) and are distributed clonally on subsets of particular cells. Not only do the innate and adaptive immune response complement each other, they are interactive in that the innate immune response influences the type of adaptive response (humoral or cellular), while the adaptive immune response can influence the function of innate immune cells. For example, the mannose receptor is a pattern-recognition molecule expressed on macrophages and DCs that recognizes the mycobacterial lipoglycan lipoarabinomannan (LAM), promoting phagocytosis¹ (innate response) and antigen presentation to T cells² (adaptive response). Activated T cells, in turn, can produce interferon-γ (IFN-γ), which activates macrophages³ and enhances T-cell antigen presentation by DCs.

Toll-like receptors (TLRs) are evolutionarily conserved pattern-recognition receptors resembling the toll proteins that mediate antimicrobial responses in Drosophila.^4,5 The TLR signalling pathway has been dissected using in vitro models of LPS induction.^6,7 LPS binds with high affinity to CD14, which leads to activation of TLR4, initiating an intracellular signalling cascade resembling that of the interleukin (IL)-1 receptor.^6,7 Activation of TLRs can also lead to induction of down-regulatory cytokines, such as IL-10,⁸ and host cell apoptosis,⁹ contributing to tissue injury. Under pathophysiological conditions, activation may lead to septic shock. Regulation of this signalling pathway is therefore vital to modulating innate immunity.

We have previously found that TLR2 is a pattern-recognition receptor for microbial lipoproteins, specifically the Mycobacterium tuberculosis 19-kDa lipoprotein, Borrelia burgdorferi OspA lipoprotein and the 47-kDa lipoprotein of Treponema pallidum.¹⁰ Bacterial lipoproteins elicit a TLR2-dependent microbicidal activity from macrophages¹¹ and can influence adaptive immune responses by triggering IL-12 production in macrophages through TLR2.¹⁰ TLR2 also mediates the recognition of other microbial patterns from M. tuberculosis, including lipoarabinomannan.¹² Studies using transfected cell lines indicate that TLR2 forms heterodimers with TLR1,¹³ conferring unique ligand-recognition patterns in comparison to TLR2 alone.¹⁴

Although several studies have demonstrated TLR expression on isolated cell populations, including peripheral blood monocytes⁷ and DCs,⁸ as well as endothelial cells,¹⁵ little is known about TLR expression patterns in intact tissue. In the current study we investigated TLR1 and TLR2 expression in lymphoid tissue as a means of understanding the role of TLRs in human immune responses. The distribution and cellular phenotype of TLR1 and TLR2 were identical. TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and DCs. TLR1 and TLR2 were also detected in the mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. Our data indicate that TLR1 and TLR2 are expressed together on myeloid cells in lymphoid tissue, providing the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.

Materials and methods

Human lymphoid tissue

Human lymphoid tissue was obtained from the Human Tissue Research Center (HTRC) at the UCLA School of Medicine with IRB approval. Biopsy specimens were embedded in OCT medium (Ames Co., Elkhart, IN) and snap-frozen in liquid nitrogen. Sections (3–5-µm thick) were acetone-fixed and kept frozen (−80°) until use.

Immunohistochemical studies of TLR expression in human lymphoid tissue

Antibodies used for immunohistochemical studies are shown in Table 1. Frozen tissue sections were blocked with normal horse serum before incubation with the monoclonal antibodies (mAbs) for 60 min, followed by incubation with biotinylated horse anti-mouse immunoglobulin G (IgG) for 30 min. Primary antibody was visualized using the ABC Elite system (Vector Laboratories, Burlingame, CA), which uses avidin and a biotin-peroxidase conjugate for signal amplification. ABC reagent was incubated for 30 min followed by the addition of substrate (3-amino-9-ethylcarbazole) for 10 min. Slides were counterstained with haematoxylin and mounted in crystal mounting medium (Biomeda, Foster City, CA). In order to enhance the detection of TLR protein expression, a tyramide signal amplification (TSA) kit was used in some cases (TSA biotin system; Perkin Elmer, Boston, MA). Western blot analysis and cell transfection studies demonstrated that the TLR1¹⁶ and TLR2 (see ref. 9 for 2392 and company insert for TLR2.3) antibodies used in this study exhibited no cross-reactivity against the other TLR protein.

Table 1.

Antibodies used to evaluate Toll-like receptor 1 (TLR1) and Toll-like receptor 2 (TLR2) expression in human lymphoid tissue

Antibody specificity	Clone	Isotype	Source
CD3	B355.1	IgG3	Biomeda
CD20	L-26	IgG2a	Dako
CD14	RPA-M1	IgG2b	Zymed
CD68	Y1/82 A	IgG2b	Phamingen
CD1a	NA1/34	IgG2a	Dako
CD1b	BCD1b3.1	IgG1	Ref. 37
TLR1	GD2.F4	IgG1	Alexis; ref. 14
TLR2	2392	IgG1	Ref. 7
TLR2	TLR2.3	IgG2a	Alexis
Cytokeratin 8	35BH11	IgM	Dako
Cytokeratin 10/13	DE-K13	IgG2a	Santa Cruz Biotechnology
Cytokeratin 14	CKB1	IgM	Sigma
Factor VIII	Polyclonal	Rabbit	Zymed

Two-colour immunofluorescence staining of cryostat sections

Double immunofluorescence was performed by serially incubating cryostat tissue sections with mouse anti-human mAbs of different isotypes [e.g. NA 1/34 (anti-CD1a, IgG2a), and 2392 (anti-TLR2, IgG1)], followed by incubation with isotype-specific, fluorochrome [fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)]-labelled goat anti-mouse immunoglobulin antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL). Controls included staining with isotype-matched antibodies, as well as with CD1a or TLR2, followed by secondary antibodies mismatched to the primary antibody isotype, to demonstrate the isotype specificity of the secondary labelled antibodies. In some cases, a weak signal was amplified using isotype-specific secondary antibodies followed by strepavidin-conjugated fluorochrome or with a TSA kit (Molecular Probes). In order to detect TLR1 and TLR2 together, a second TLR2 antibody was used (TLR2.3; mouse IgG2a) because TLR1 (GD2.F4) and TLR2 (2392) are both mouse IgG1 mAbs. Images were obtained using confocal laser microscopy.

Confocal microscopy

Double immunofluorescence of sections were examined using a Leica-TCS-SP inverted confocal laser-scanning microscope fitted with krypton and argon lasers at the Carol Moss Spivak Cell Imaging Facility in the UCLA Brain Research Institute. Sections were illuminated with 488 and 568 nm of light after filtering through an acoustic optical device. Images decorated with FITC, TRITC, Alexa 488, or Alexa 568 (Molecular Probes) were recorded simultaneously through separately optical detectors with a 530-nm band-pass filter and a 590-nm long-pass filter, respectively. Pairs of images were superimposed for co-localization analysis.

Results

TLR2 expression in lymphoid tissue

Although several studies have investigated the functional outcome of TLR2 signalling using peripheral blood monocytes and monocyte-derived DCs,^8,17,18 the in vivo expression of this pattern-recognition receptor has not been characterized in detail. We investigated the in vivo expression of TLR2 in lymphoid tissue to determine the phenotype and distribution of cells that can contribute to innate immunity. TLR2 was found in the medullary cords of lymph nodes (Fig. 1A). In the spleen (Fig. 1B), TLR2 was expressed in the marginal zone and splenic cords of the red pulp, where phagocytic macrophages are located. TLR2-positive cells were found in the corticomedullary junction of thymus (Fig. 1C) and in the medulla of tonsil (Fig. 1D). The highest level of expression (i.e. greatest frequency of cells) was detected in spleen. The distribution of TLR2-positive cells correlated with the expression of distribution of CD14⁺ monocytes and CD1a⁺ DCs (Fig. 1A−1D), suggesting that TLR2 is expressed on macrophages and DCs in lymphoid tissue, as has been previously shown for peripheral blood.

Figure 1.

Toll-like receptor 2 (TLR2) expression in human lymphoid tissue. Lymphoid tissue was labelled with monoclonal antibodies (mAbs) specific for TLR2, CD14 and CD1a using the immunoperoxidase method. Original magnification 100× (panels a, c and d); 400× (b). The box in panel (a) indicates the section magnified in panel (b). (A) Human lymph nodes labelled with mAbs specific for TLR2⁺ cells are located in the medullary cords (M) of the lymph nodes. Macrophages (CD14⁺) and dendritic cells (DCs) (CD1a⁺) localized to the same region as TLR2-positive cells TLR2 expression was weak to negative in follicles (F). (B) TLR2-expressing cells in human spleen are located predominantly at the splenic cords and at the interface between the white pulp (WP) and the red pulp (RP) or marginal zone (MZ), the same site as macrophages and DCs. (C) TLR2-positive cells in thymus are found predominantly at the medullary junction (MJ) with a limited number located at the cortex (C) and the medulla (M). Macrophages and DCs are scattered throughout the medulla, cortex and medullary junction. CD1a⁺ thymocytes are abundant in the cortex. (D) TLR2-positive cells in tonsil are localized in the medullary area, as are macrophages and DCs.

TLR2 co-localizes with macrophage and DC markers

To determine the phenotype of TLR2-positive cells in lymphoid tissue, frozen tissue sections were labelled with antibodies to macrophage, DC, and lymphocyte markers simultaneously with anti-TLR2 antibody. TLR2 co-localized with CD1a⁺ DCs and CD14⁺ macrophages in tonsil (Fig. 2). Similar co-localization was seen with spleen and lymph node (data not shown). In contrast, although TLR2 cells were adjacent to lymphocytes, T and B cells did not express TLR2 (Fig. 2). These data indicate that TLR2 expression is restricted to cells of the innate immune system in human lymphoid tissue.

Figure 2.

Toll-like receptor 2 (TLR2) expression on monocytes and dendritic cells (DCs) in human lymphoid tissue. TLR2-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR2-positive cells expressed markers for DCs (CD1a) and macrophages (CD68 and CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (center column) are TLR2 and superimposed images are shown in right column. Magnification 630×. The frequency of TLR2⁺ cells in lymphoid tissue is greater than the number of macrophages or DCs in situ as both macrophages and DCs populate lymphoid tissues and therefore the number of TLR2⁺ cells should be no less than the sum of CD14⁺ and CD1a⁺ cells.

TLR1 is expressed on monocytes and DCs and co-localizes with TLR2

TLR1 forms heterodimers with TLR2 in the cytoplasm of TLR2-expressing cells.¹³ These heterodimers confer distinct microbial pattern recognition in comparison to either TLR alone.¹⁴ We reasoned that TLR2 and TLR1 should be expressed on the same cells and thus we examined the expression of TLR1 and TLR2 in lymphoid tissue using immunofluorescence. TLR1 was expressed in the medullary area of lymphoid tissue (Fig. 3A) and co-localized with macrophage and DCs markers, but not lymphocyte markers, similarly to TLR2 (Fig. 3B). TLR1 also co-localized with TLR2 on cells in lymphoid tissue (Fig. 3C). As TLR1 was detected only on monocytes and DCs, we conclude that TLR1 and TLR2 are co-expressed on myeloid cells of the innate immune system in lymphoid tissue.

Figure 3.

Toll-like receptor 1 (TLR1) expression on monocytes and dendritic cells (DCs) in lymphoid tissue. (A) Human tonsils labelled with monoclonal antibodies (mAbs) specific for TLR1 (a, b). TLR1-positive cells in tonsil localized to the medulla. Original magnification 200× (a) and 400× (b). The box in panel (a) indicates the region magnified in panel (b). (B) TLR1-expressing cells were characterized for cell lineage in human tonsil using two-colour immunofluorescence confocal laser microscopy. TLR1-positive cells expressed markers for DCs (CD1a) and macrophages (CD14), but did not express T-cell (CD3) or B-cell (CD20) markers. Green images (left column) are CD markers, red images (centre column) are TLR1 and superimposed images are shown in the right column. Original magnification 630×. An arrow denotes the CD14⁺ cell in the center of the panel. (C) TLR1 co-localizes with TLR2 in lymphoid tissue. TLR1 (green) and TLR2 (red) were labelled and detected as described above (Fig. 2) and superimposed to identify co-localization.

TLR2 is expressed on epithelial cells in mucosa-associated lymphoid tissue

Epithelial cells of mucosa-associated lymphoid tissues have immune surveillance functions, as they interact directly with the environment. Although human spleen and lymph nodes expressed TLR2 exclusively on macrophages and DCs, TLR2 expression was also detected on the squamous epithelium of tonsillar crypts (Fig. 4A). To confirm that the cells expressing TLR2 were the crypt epithelial cells, we examined the expression of several cytokeratin markers for epithelial cells. TLR2-expressing cells co-localized with a subset of cytokeratin 10/13, but not with cytokeratin 8 or 14 (Fig. 4B), confirming that crypt epithelial cells express TLR2. We also found that TLR2 was not expressed on endothelial cells in vivo (Fig. 4C), as previously described in vitro.¹⁵ These data suggest that epithelial cells of the mucosa-associated lymphoid tissue may be involved in the innate immune response to bacterial lipoproteins. TLR1 was also expressed on tonsillar epithelium with the same expression pattern as TLR2 (data not shown).

Figure 4.

Toll-like receptor 2 (TLR2) expression on epithelial cells in lymphoid tissue. (A) Immunoperoxidase staining of sections demonstrates that TLR2-positive cells are also distributed throughout the tonsillar crypt epithelium (TCE). Original magnification 100× (a) and 200× (b). (B) Double-immunofluorescence labelling of tonsil sections with cytokeratin (CK) 8, 14 and 10/13 (green; left panel of each row) and TLR2 (red; centre panel of each row) demonstrate co-localization of TLR2 with CK10/13, but not CK8 or CK14. Original magnification 630×. (C) Double-immunofluorescence labelling with endothelial cell marker Factor VIII (green; first panel) and TLR2 (red) illustrates a lack of co-localization (right panel).

Discussion

Cells of the innate immune system express receptors that recognize and respond to the molecular patterns of microbial pathogens. We investigated the pattern of TLR1 and TLR2 expression in human lymphoid tissue to determine how distinct receptors of the immune system can contribute to immune responses. TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and DCs. In addition, TLR1 and TLR2 were expressed in the mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, the lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.

In the current study, we found TLR2 to be expressed on macrophages in secondary lymphoid tissue, including lymph node, tonsil and spleen. Activation of TLRs on macrophages leads to the transcription of genes, expression of proteins and functional changes that influence both innate and adaptive immune responses. For example, TLR activation affects the ability of macrophages to phagocytose foreign material and release pro-inflammatory cytokines that contribute to innate immunity.^8,19 We recently found that TLR2 activation leads to the killing of intracellular M. tuberculosis in both mouse and human macrophages, although through distinct mechanisms.¹¹ Some of these antimicrobial activities may take place within phagocytic vesicles, where TLR agonists, such as the 19-kDa lipoprotein of M. tuberculosis, can co-localize with TLRs.^13,20 These findings suggest that TLR2 expression on macrophages (and perhaps in phagosomal compartments) allows detection of an invader and induction of appropriate antimicrobial responses.

We also found TLR1 and TLR2 expression on DCs, potent stimulators of adaptive immune responses. During the evolution of an immune response, DCs encounter antigen in the periphery and migrate via lymphatics to the lymph node where they activate T cells to proliferate and differentiate into effector cells. Two functional subsets of DCs have been described from peripheral blood ex vivo that express distinct patterns of TLRs. CD11c⁺ DCs induce T helper 1 (Th1) adaptive immune responses, whereas plasmacytoid DCs induce T helper 2 (Th2) responses.²¹ TLR1 and TLR2, but not TLR9, are expressed on CD11c⁺ DCs;^22,23 in contrast, plasmacytoid DCs express TLR9, but not TLR2 and relatively low levels of TLR1.^22,23 Therefore, we conclude that TLR1- and TLR2-positive DCs in lymphoid tissue are CD11c⁺ DCs. TLR1 and TLR2 expression on DCs and macrophages in secondary lymphoid tissue in close proximity to T cells suggests that they are functionally capable of modulating adaptive immune responses in vivo. One can envision a situation where TLR activation leads to maturation of DCs,^17,18 resulting in more efficient antigen presentation, mediated by the up-regulation of costimulatory proteins²⁴ and increased major histocompatibility complex (MHC) class II expression.²⁵ In addition, TLR-induced cytokines that influence a T-helper phenotype^8,10 would be produced at the initiation site of an adaptive immune response.

TLR1 and TLR2 were also expressed on epithelial cells of the tonsil, suggesting that epithelial cells can recognize and respond to microbial patterns. As a lymphoepithelial organ, tonsil has an immune surveillance function distinguishing it from lymph nodes and spleen amongst secondary lymphoid tissue. Therefore, it is not surprising to find pattern-recognition receptors, such as TLRs, on tonsillar epithelium. Epithelial cells of mucosa-associated lymphoid tissue can produce antimicrobial factors²⁶ and cytokines,^27,28 and can present antigen to T cells.^29,30 Our findings therefore suggest that by expressing TLRs, epithelial cells can sample molecular patterns and have direct antimicrobial functions and influence adaptive immune responses. TLR2 was found on a subset of epithelial cells, supporting the hypothesis that responsiveness to distinct microbial patterns is preferred in certain in situ environments.¹⁵

Lymphocytes were devoid of TLR1 and TLR2 expression in secondary lymphoid tissue. This is not surprising because T cells generally do not directly respond to lipoproteins (ref. ³¹, and our own unpublished observations). Lipoproteins are only weak mitogens for human B cells,^31,32 supporting the finding that TLR2 was undetectable on B cells in vivo. The weak response of human B cells to lipoproteins may be explained if only a minor subset of B cells in lymphoid tissue respond to lipoprotein, or B cells require an additional signal to respond to lipoprotein.³² An alternative explanation for the ability of human B cells to respond to lipoprotein in the absence of detectable TLR2 is that another TLR family member confers lipoprotein responsiveness on B cells, similar to the effect of RP-105 on the LPS responsiveness of B cells.^33,34

As ligand–receptor relationships are identified for TLRs, a paradigm is developing, suggesting that some TLRs function as primary receptors whereas others are accessory proteins that confer alternative recognition patterns on innate immune cells. That is, TLRs form heterodimers, generating combinatorial diversity to increase the range of microbial ligand recognition. TLR6, in combination with TLR2, is required for recognition of mycoplasmal lipoprotein, which contains a diacylated N-terminal cysteine, whereas recognition of the triacylated cysteine of Gram-positive and Gram-negative lipoproteins requires TLR2, but does not have a requirement for TLR6.³⁵ In contrast, when TLR2 combines with TLR1, a novel pattern-recognition receptor is formed that recognizes triacylated lipoproteins³⁶ as well as a soluble factor released by the Gram-negative bacterium Neisseria meningitidis.¹⁴ We found that TLR1 and TLR2 are expressed together on macrophages and DCs, supporting the hypothesis that they function together. Our study demonstrates that macrophages and DCs in lymphoid tissue are equipped to respond to Gram-positive (TLR2; see ref. 10) and Gram-negative bacteria (TLR1 combined with TLR2; see ref. 14) via TLR expression. The expression of TLR1 and TLR2 on macrophages and DCs in lymphoid tissue suggests that therapies that target these cells could help engender immune responses in the treatment of infection and cancer.