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Original Article |

Immunohistological Expression of Interleukin 16 in Human Tonsils FREE

Matthias F. Kramer, MD; Brigitte Mack; Gerd Rasp, MD
[+] Author Affiliations

From the Department of Oto-Rhino-Laryngology/Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.


Arch Otolaryngol Head Neck Surg. 2001;127(9):1120-1125. doi:10.1001/archotol.127.9.1120.
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Published online

Background  Interleukin 16 (IL-16) acts highly chemotactic on CD4-bearing cells. Besides chemotaxis, IL-16 has numerous immunomodulatory effects, and not only on T cells.

Objective  To determine IL-16 expression in human tonsils.

Methods  Tonsillar follicles were immunohistologically characterized to elicit a possible cellular source of IL-16 expression.

Results  The mantle zone of immature and mature B cells was CD22 immunoreactive (ir), whereas the germinal center of activated B cells was CD23-ir. Plasma cells that were CD38-ir were observed extrafollicularly beneath the epithelium and within the germinal center. T cells were found most frequently in the extrafollicular space, with a majority of CD4 cells. CD68-ir macrophages were predominantly found within the germinal center. Immunostaining of anti–IL-16 revealed strong cytoplasmatic reactivity of extrafollicular cells and of cells at the outer rim of the mantle zone. Numerous cells adherent to the stratified squamous epithelium were IL-16-ir as well. Double immunostaining identified CD4+ T cells as the major cellular source of IL-16 expression. Furthermore, a population of CD22+ B cells at the outer rim of the mantle zone expressed IL-16 as well.

Conclusions  Interleukin 16 was mainly expressed in a typical CD4-like pattern in human tonsils. Our data strongly suggest that CD4+ lymphocytes constitute the major cellular source for IL-16. We hypothesize that the double-immunostained CD4-ir and IL-16-ir cells represent activated T cells. Because CD22+ B cells at the outer rim of the mantle zone expressed IL-16 as well, we conclude that this area might constitute the locus of IL-16–mediated B-cell differentiation.

Figures in this Article

INTERLEUKIN 16 (IL-16) is synthesized as a precursor molecule of 68 kd (pro–IL-16) that is processed by caspase-3. The cleavage results in a 13-kd carboxy terminal peptide, which constitutes the bioactive form of IL-16.1 Interleukin 16, a proinflammatory and immunomodulatory cytokine formerly known as lymphocyte chemoattractant factor, acts highly chemotactic on CD4-bearing cells. Effects of IL-16 are transmitted by its surface receptor CD4.2

Effects of IL-16 on CD4+ T cells include chemotaxis, cell adhesion, induction of HLA-DR, induction of cytokine synthesis, and induction of IL-2 receptor expression (CD25).24 Furthermore, IL-16 is known as a CD4+ T-cell growth factor because it is capable of inducing a G0 to G1 cell cycle change.3 The cumulative effects of these functions on CD4+ T cells are increased CD4+ cell recruitment, priming for IL-2–responsive proliferation, and protection against Fas-mediated apoptosis.2,3

Besides affecting T cells, IL-16 also acts on other inflammatory cells. It is chemotactic on eosinophils and monocytes and induces HLA-DR expression in the latter.2 Furthermore, IL-16 might constitute an important mediator in cell-cell interactions of various inflammatory cells such as dendritic, mast, or B cells.58

Most work to date has focused on the chemotactic activities of IL-16 and therefore on diseases characterized by tissue infiltration of CD4+ cells. In individuals with atopic asthma, IL-16 represents a major source of lymphocyte chemotactic activity early after antigen challenge in which the major cell of origin is the epithelium, although mast cells, CD8 and CD4 cells, and eosinophils were described as additional sources.9 Kaser and coworkers,8 on the other hand, suggested that activated T cells are a cellular source of IL-16 expression. Dendritic cells were described recently as another source of IL-16.8

Interleukin 16 is released from CD4+ T cells in response to antigen, mitogen, histamine, or anti-CD3 stimulation. Interleukin 16 messenger RNA and pro–IL-16 are constitutively expressed by CD4+ and CD8+ T cells. But different from CD8+ T cells, processing and release of bioactive IL-16 by CD4+T cells is activation dependent.1

An increased level of IL-16 after challenge has been described in asthma9,10 and in allergic rhinitis,11 in which IL-16 expression correlated with CD4 influx. Recently, Kramer et al12 identified IL-16 as a characteristic cytokine expressed in late-phase response after antigen challenge in allergic rhinitis. Increased expression of IL-16 was demonstrated in other chronic inflammatory processes characterized by CD4+ T-cell influx, such as atopic dermatitis, or granulomatous inflammation, such as sarcoidosis or tuberculosis,2,13 multiple sclerosis,14 and acquired immunodeficiency syndrome.15 Even autoimmune diseases such as systemic lupus erythematosus are characterized by increased levels of IL-16, which correlate with disease activity.16

To our knowledge, IL-16 expression by tonsillar tissue has not been studied to date. Tonsillar follicles belong to the organized mucosa-associated lymphoid tissue of the Waldeyer ring.17 Their B-cell and T-cell areas are the locus of lymphocyte differentiation and initiation of mucosal immune responses. Intense B-cell– T-cell interactions take place, suggesting a relevant role for IL-16 here.

Tonsils are locations of intense cell-cell interactions while promoting immune responses. As described in the previous paragraphs, IL-16 has far more immunomodulatory effects on various inflammatory cells than chemotaxis.

The aim of this study was to determine IL-16 expression in human tonsils. Tonsillar follicles were immunohistologically characterized to elicit a possible cellular source of IL-16 expression.

Tonsils were obtained from 5 patients (mean ± SD age, 6.4 ± 1.4 years) undergoing routine tonsillectomy for recurrent tonsillitis without acute exacerbation. An allergic condition was ruled out by in vivo (skin test) and in vitro (Sx1 [identical to Phadiatop]; Pharmacia & Upjohn, Freiburg, Germany) tests to exclude possible effects of an atopic disease.

Tonsillar follicles were immunohistologically characterized to elicit a possible cellular source of IL-16 expression. B-cell differentiation and activation occur during migration from the outer mantle zone toward the germinal center.5 As a marker for immature and mature B cells, we chose anti-CD22, as described previously.18 Expression of CD23 in B cells is associated with immunoglobulin isotope switching. Therefore, CD23+ B cells are generally accepted to constitute activated B cells.18 Anti-CD38 was taken for detection of plasma cells.19 T-cell populations were detected by anti-CD3 (pan T-cell marker), and T-cell subpopulations were identified by anti-CD4 and anti-CD8. For detection of macrophages we chose anti-CD68 (clone KP1), as described elsewhere.20

IMMUNOHISTOCHEMISTRY

Specimens were shock frozen in liquid nitrogen and stored at –20°C. Cryostat sections (5 µm) were postfixed with acetone for 10 minutes. Thereafter, sections were rinsed in 0.05M phosphate-buffered saline solution (pH 7.4). Endogenous peroxidases were blocked by incubation in 0.3% hydrogen peroxide for 30 minutes. Nonspecific binding was minimized by incubation with fetal calf serum (1:20) for 20 minutes. The avidin-biotin-peroxidase complex method was used for detection of immunostaining, as described elsewhere.21 Briefly, the following antibodies were used: monoclonal (mc) anti-CD3 (1:200), mc anti-CD4 (1:100), mc anti-CD8 (1:200), mc anti-CD22 (1:100), mc anti-CD23 (1:3000), mc anti-CD38 (1:2000), mc anti-CD68 (clone KP1, 1:10 000, all mouse) (Dako, Glostrup, Denmark), and polyclonal anti–IL-16 (1:100, goat) (R&D, Wiesbaden, Germany). Slides were incubated with the primary antibody for 1 hour. After washing, slides were incubated with a biotinylated secondary antibody for 30 minutes (1:200) (Vector Lab, Burlingame, Calif). An avidin-biotin-peroxidase complex (Vector Lab) was added for 30 minutes after washing. Finally, peroxidase reaction was performed using 0.01% 3-amino-9-ethylcarbazole (Sigma, Munich, Germany) as chromogen. Omission of the primary antibody abolished the immunohistological staining completely. Haemalum staining was performed as the last step of the procedure to counterstain the nuclei.

A "Zeiss Standard 25" light microscope (Carl Zeiss, Oberkochen, Germany) was used. Photographs were taken on Kodak 160T film (Eastman Kodak, Rochester, NY).

DOUBLE IMMUNOSTAINING

Double immunostaining was performed with anti–IL-16 using the avidin-biotin-peroxidase complex (red) and alkalic phosphatase–antialkalic phosphatase (blue) methods for the other previously mentioned antibodies as described elsewhere.22 The avidin-biotin-peroxidase complex method was carried out as described in the previous subsection. Briefly, the alkalic phosphatase–antialkalic phosphatase method was carried out via incubation of the primary antibody. A rabbit anti–mouse immunoglobulin antibody (1:25) (Dako) was used as the secondary antibody. A 0.05M Tris-buffered saline solution (pH 7.6) was taken instead of phosphate-buffered saline solution as washing solution. Alkalic phosphatase–antialkalic phosphatase complex (1:50) (Dako) was added thereafter. Fast Blue BB salt (Dako) was used as the final staining substrate.

B-CELL AREA

The mantle zone of immature and mature B cells was CD22 immunoreactive (ir), whereas the germinal center of activated B cells was CD23-ir. CD38-ir plasma cells were observed extrafollicularly beneath the epithelium and within the germinal center (Figure 1, Figure 2, and Figure 3).

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Figure 1.

Immature and mature B cells of the mantle zone of the tonsillar secondary lymph follicle are CD22 immunoreactive (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 2.

Staining by anti-CD23 detected activated B cells in the germinal center of the tonsillar secondary lymph follicle (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×100).

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Figure 3.

Anti–CD38 antibody–stained plasma cells. Plasma cells were found extrafollicularly beneath the epithelium and, as shown here, within the germinal center of the follicle (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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T-CELL AREA

T cells (CD3+) were found most frequently in the extrafollicular space, with a majority of CD4 cells. CD4-ir lymphocytes were observed in the mantle zone of the follicle too, whereas CD4-ir cells were only occasionally found in the germinal center (Figure 4, Figure 5, and Figure 6).

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Figure 4.

CD3+ T cells were found in the extrafollicular space of the tonsillar tissue. Diapedesis of the stratified squamous epithelium by CD3+ T cells is shown (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 5.

Staining by anti-CD4 revealed T-helper cells mainly in the extrafollicular space. Note few CD4+ T cells within the mantle zone and the germinal center (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 6.

CD8+ T cells were basically restricted to the extrafollicular space. Comparing anti-CD8 with anti-CD4 staining showed that most T cells are CD4 immunoreactive (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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MACROPHAGES

CD68-ir macrophages were inhomogeneously distributed within the tonsillar tissue, with a predominance in the germinal center (Figure 7).

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Figure 7.

Double immunostaining with anti-CD68 (clone KP1, blue) and anti–interleukin 16 (red) revealed an inhomogeneous distribution of macrophages, with a predominance in the germinal center (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; ×200).

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EPITHELIUM

Invaginated stratified squamous epithelium formed tonsillar crypts. Diapedesis of the epithelium by CD4-ir and CD8-ir cells was observed. Cells adherent to the epithelium were CD4-ir and could be detected regularly.

IL-16 IMMUNOSTAINING

Immunostaining of anti–IL-16 revealed strong cytoplasmatic reactivity of extrafollicular cells and of cells at the outer rim of the neighboring mantle zone. Only a few cells within the germinal center were IL-16-ir (Figure 8). Several cells adherent to the stratified squamous epithelium were IL-16-ir as well.

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Figure 8.

Interleukin 16 (IL-16)–immunoreactive cells were found mainly extrafollicularly and within the mantle zone of tonsillar secondary lymph follicle. Only a few cells within the germinal center were IL-16+ as well (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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DOUBLE STAINING

Double immunostaining identified CD4+ T cells as the major cellular source of IL-16 expression. Most IL-16-ir cells were also CD4-ir, but not all CD4-ir T cells expressed IL-16. Furthermore, a population of CD22+ B cells at the outer rim of the mantle zone expressed IL-16 as well (Figure 9, Figure 10, and Figure 11).

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Figure 9.

Double immunostaining with anti-CD4 (blue) and anti–interleukin 16 (IL-16) (red) showed that the pattern of IL-16 expression is similar to the expression of CD4+ T cells. Most cells within the extrafollicular space appear double stained (dark brown). These IL-16 and CD4-immunoreactive cells are suggested to constitute activated T cells (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×200).

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Figure 10.

Double immunostaining with anti-CD4 (blue) and anti–interleukin 16 (IL-16) (red) at a high magnification. Most cells within the extrafollicular space appear double stained (dark brown). Note that within the extrafollicular space no cells other than CD4+ express IL-16, whereas not all CD4-immunoreactive T cells express IL-16. Interleukin 16 and CD4 double-immunoreactive cells are suggested to constitute activated T cells (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×400).

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Figure 11.

Double immunostaining with anti-CD22 (blue) and anti–interleukin 16 (IL-16) (red) at a high concentration. Presented is the mantle zone of a tonsillar secondary lymph follicle. Extrafollicular cells at the upper parts of the figure are IL-16 immunoreactive (red), whereas B cells of the mantle zone were CD22 immunoreactive (blue, compare with Figure 1). Note the double-stained cells (dark brown) at the outer rim of the mantle zone. This zone is suggested to constitute the locus of IL-16–mediated B-cell differentiation (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×400).

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Immunohistological characterization of human tonsillar follicles displayed the well-known structure of secondary lymph follicles: The mantle zone of immature and mature B cells was CD22-ir, whereas the germinal center of activated B cells was CD23-ir. The precursors of germinal center B cells are believed to be mantle B lymphocytes.5 Germinal centers represent antigen-dependent B-cell compartments responsible for proliferative expansion of memory clones and differentiation to immunoglobulin-producing immunocytes.5 CD38-ir plasma cells were observed extrafollicularly beneath the epithelium and within the germinal center. T cells were found most frequently in the extrafollicular space. CD4+ cells were dominant. CD4-ir lymphocytes were observed in the mantle zone of the follicle too, whereas CD4-ir cells were only occasionally found in the germinal center. CD68-ir macrophages were inhomogeneously distributed within the tonsillar tissue, with a predominance in the germinal center. All this is in accordance with results of other researchers5,18 who described human tonsillar follicles using immunohistochemical analysis.

Immunostaining of anti–IL-16 revealed strong cytoplasmatic reactivity of extrafollicular cells and of cells at the outer rim of the neighboring mantle zone. Only a few cells within the germinal center were IL-16-ir. Several cells adherent to the stratified squamous epithelium were IL-16-ir too. Double immunostaining identified CD4+ T cells as the major cellular source of IL-16 expression. Most IL-16-ir cells were also CD4-ir, but not all CD4-ir T cells expressed IL-16. Furthermore, a population of CD22+ B cells at the outer rim of the mantle zone expressed IL-16 as well. This immunohistological distribution of IL-16-ir CD4+ T cells and IL-16-ir CD22+ B cells suggests that the outer rim of the mantle zone constitutes the location of IL-16–mediated B-cell differentiation.

Immunohistological examination of IL-16 expression in human lymphoid organs other than tonsils revealed similar results: examination of human lymph nodes showed that IL-16 is immunohistologically expressed in lymphocytic cytoplasm within T-cell zones extrafollicularly and only occasionally in lymphocytes of the germinal center.23

Interleukin 16 was mainly expressed in a typical CD4-like pattern in human tonsils. Unlike atopic airways, our data strongly suggest that CD4+ lymphocytes constitute the major cellular source of IL-16 in human tonsils. Furthermore, and unlike atopic airways, CD8+ T cells, the epithelium, or macrophages did not express IL-16, at least not by means of immunohistochemistry. In addition, we found IL-16-ir CD22+ B cells. These B cells might contribute to the observed IL-16 expression as well.

Double immunostaining clearly identified CD4+ cells as the major cellular source of IL-16 expression. Comparison with anti-CD3 staining led us to believe that most of these CD4+cells are T cells. However, CD4-bearing cells other than lymphocytes under this IL-16-ir cell population cannot be excluded in the end but seem unlikely to be of relevant amounts.

Tonsillar T cells are suggested to enter the extrafollicular tonsillar area through so-called postcapillary or high-endothelial venules from the blood.5 Their extravasation and chemoattractance are not understood. αβ-Leukointegrins are suggested to constitute selective adhesion receptors.24 Our data suggest chemotactic effects of IL-16 on these CD4+ T cells.

As described earlier in this article, IL-16 has far more immunomodulatory effects on various inflammatory cells than chemotaxis. Interleukin 16 further promotes cell adhesion, HLA-DR induction, and induction of cytokine synthesis on T cells and acts as a T-cell growth factor.2,3 Induction of IL-2 receptor expression (CD25) is mediated by IL-16 as well, leading to a synergistic activation of CD4+ T cells by IL-16 and IL-2.4 The cumulative effects of these functions on CD4+ T cells are increased CD4+ cell recruitment and priming for IL-2–responsive proliferation.2,3 Tonsillar T lymphocytes have striking peculiarities: most are activated, express IL-2 receptor (anti-Tac positive), and respond to IL-2 stimulation.5,25 Furthermore, most of these activated T lymphocytes are T-helper cells5,26 and are localized mainly extrafollicularly.27 Results of in vitro studies5 have indicated that these IL-2–activated T cells are involved in differentiation of tonsillar B cells to plasma cells, which is indirectly supported by our data. Furthermore, tonsillar B and T cells are characterized by more frequent DR expression compared with blood lymphocytes.5 The abundant intrafollicular DR expression (B cells, T cells, dendritic cells, etc) probably serves to modulate local interactions between antigen-presenting cells, T cells, and B cells.5 Taken together, our data point to IL-16 as a relevant factor in the immune response of human tonsils. Different from CD8+ T cells, processing and release of bioactive IL-16 by CD4+T cells is activation dependent.1 Therefore, our data might be interpreted as showing that the observed IL-16-ir and CD4-ir cells constitute activated T cells. Triple staining with IL-2 receptor promises to be a challenging but interesting target for a future study.

Besides affecting T cells, IL-16 acts on other inflammatory cells too. It is chemotactic for eosinophils and monocytes and induces HLA-DR expression in the latter.2 Furthermore, IL-16 constitutes an important mediator in cell-cell interactions of various inflammatory cells: T cells and mast cells interact bidirectionally in various aspects of immune response, in which IL-16 plays an important role,7 and T-cell–dendritic cell interactions also depend on IL-16.8 Interleukin 16 is important in B-cell–T-cell interactions as well. For instance, B-cell precursors need IL-16 for their maturation,6 which again is supported by our finding of IL-16-ir CD22+ B cells. All these interactions are pivotal parts in initiating and promoting an immune response and point to IL-16 as a relevant factor.

Interleukin 16 is mainly expressed in a typical CD4-like pattern in human tonsils. Our data strongly suggest that CD4+ lymphocytes constitute the major cellular source for IL-16. We hypothesize that the double-immunostained CD4-ir and IL-16-ir cells represent activated T cells. CD22+ B cells at the outer rim of the mantle zone expressed IL-16 as well, leading us to conclude that this area might constitute a locus of IL-16–mediated B-cell differentiation.

Accepted for publication February 7, 2001.

Corresponding author and reprints: Matthias F. Kramer, MD, Department of Oto-Rhino-Laryngology/Head and Neck Surgery, Ludwig-Maximilians-University, Klinikum Grosshadern, Marchioninistr.15, 81377 Munich, Germany (e-mail: mkramer@hno.med.uni-muenchen.de).

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Figures

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Figure 1.

Immature and mature B cells of the mantle zone of the tonsillar secondary lymph follicle are CD22 immunoreactive (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 2.

Staining by anti-CD23 detected activated B cells in the germinal center of the tonsillar secondary lymph follicle (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×100).

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Figure 3.

Anti–CD38 antibody–stained plasma cells. Plasma cells were found extrafollicularly beneath the epithelium and, as shown here, within the germinal center of the follicle (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 4.

CD3+ T cells were found in the extrafollicular space of the tonsillar tissue. Diapedesis of the stratified squamous epithelium by CD3+ T cells is shown (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 5.

Staining by anti-CD4 revealed T-helper cells mainly in the extrafollicular space. Note few CD4+ T cells within the mantle zone and the germinal center (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 6.

CD8+ T cells were basically restricted to the extrafollicular space. Comparing anti-CD8 with anti-CD4 staining showed that most T cells are CD4 immunoreactive (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 7.

Double immunostaining with anti-CD68 (clone KP1, blue) and anti–interleukin 16 (red) revealed an inhomogeneous distribution of macrophages, with a predominance in the germinal center (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; ×200).

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Figure 8.

Interleukin 16 (IL-16)–immunoreactive cells were found mainly extrafollicularly and within the mantle zone of tonsillar secondary lymph follicle. Only a few cells within the germinal center were IL-16+ as well (avidin-biotin-peroxidase complex method, Haemalum counterstain, ×200).

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Figure 9.

Double immunostaining with anti-CD4 (blue) and anti–interleukin 16 (IL-16) (red) showed that the pattern of IL-16 expression is similar to the expression of CD4+ T cells. Most cells within the extrafollicular space appear double stained (dark brown). These IL-16 and CD4-immunoreactive cells are suggested to constitute activated T cells (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×200).

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Figure 10.

Double immunostaining with anti-CD4 (blue) and anti–interleukin 16 (IL-16) (red) at a high magnification. Most cells within the extrafollicular space appear double stained (dark brown). Note that within the extrafollicular space no cells other than CD4+ express IL-16, whereas not all CD4-immunoreactive T cells express IL-16. Interleukin 16 and CD4 double-immunoreactive cells are suggested to constitute activated T cells (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×400).

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Figure 11.

Double immunostaining with anti-CD22 (blue) and anti–interleukin 16 (IL-16) (red) at a high concentration. Presented is the mantle zone of a tonsillar secondary lymph follicle. Extrafollicular cells at the upper parts of the figure are IL-16 immunoreactive (red), whereas B cells of the mantle zone were CD22 immunoreactive (blue, compare with Figure 1). Note the double-stained cells (dark brown) at the outer rim of the mantle zone. This zone is suggested to constitute the locus of IL-16–mediated B-cell differentiation (avidin-biotin-peroxidase complex: red; alkalic phosphatase–antialkalic phosphatase: blue; double stained: dark brown; ×400).

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