Adenoids provide a microenvironment for the generation of CD4+, CD45RO+, L-selectin, CXCR4+, CCR5+ T lymphocytes, a lymphocyte phenotype found in the middle ear effusion
Petri S. Mattila,
Antti Nykänen,
Marjo Eloranta1 and
Jussi Tarkkanen1
Department of Otorhinolaryngology, Helsinki University Central Hospital, Haartmaninkatu 4 E, 00290 Helsinki, Finland
1 Department of Pathology, Haartman Institute, Helsinki University Central Hospital, PO Box 21, 00014 Helsinki, Finland
Correspondence to:
P. S. Mattila
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Abstract
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Adenoidectomy in children with otitis media with effusion reduces inflammation in the middle ear by an unknown mechanism. Potentially, the adenoids of these children may serve as a site for the differentiation of lymphocytes, which after entering blood circulation eventually extravasate in the middle ear mucosa and thereby contribute to excessive inflammation. During lymphocyte extravasation various adhesion molecules and chemokines play a crucial role. To evaluate possible connections between the adenoids and middle ear inflammation, the expression of the chemokine receptors CXCR4 and CCR5 and the lymphocyte homing receptor L-selectin were analyzed in adenoidal and middle ear lymphocytes. It was found that most CD4+ T lymphocytes in the middle ear effusion express the memory phenotype marker CD45RO and the chemokine receptors CXCR4 and CCR5, but are negative for the lymphocyte homing receptor L-selectin. This cell phenotype was rare in peripheral blood but was found much more frequently in the adenoids. The results suggest that the adenoids provide a microenvironment for the generation for CD4+, CD45RO+, L-selectin, CXCR4+ and CCR5+ T lymphocytes. Further, these cells may include cells that have the capacity to home to the middle ear mucosa. As the adenoidal CD4+ memory phenotype CD45RO+ T cells expressed the activation antigen CD69 and included cells expressing the HIV co-receptors CXCR4 and CCR5 at a high level, they may be permissive for HIV infection.
Keywords: chemokine receptors, lymphocyte homing receptors, otitis media with effusion
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Introduction
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Acute otitis media is characterized by collection of effusion in the middle ear cavity. Usually the effusion and inflammation in the middle ear mucosa resolve uneventfully, but sometimes acute otitis is followed by frequent recurrencies or by persistent (>3 months) collection of effusion, also called otitis media with effusion (OME). Removal of the adenoids (adenoidectomy) has been reported to be beneficial for the treatment of children with OME (1,2). However, the mechanisms that mediate the beneficial actions of adenoidectomy in OME are unknown.
It has been postulated that enlarged adenoids obstruct the orifices of the Eustachian tubes, which provide the anatomic connection between the nasopharynx and the middle ear cavity, thereby obstructing the ventilation of the middle ear. However, no association has been found between the size of the adenoids and the prevalence of otitis media (3,4). It has also been proposed that the adenoids would provide a reservoir for pathogenic bacteria in the nasopharynx of children with OME. However, quantitative bacterial analyses of adenoid tissue from such children have failed to demonstrate any bacterial overgrowth (57).
Secondary lymphoid organs such as the adenoids provide a microenvironment for antigen-recognition of naive CD4+ T lymphocytes and differentiation to foreign antigen-experienced T lymphocytes (8). During the differentiation process, antigen-specific T cells clonally expand and gain a distinct adhesion molecule, chemokine receptor and cytokine secretion pattern, which provides the specific functional characteristics of each antigen-specific T lymphocyte clone.
Adhesion molecules, such as selectins and integrins (8,9), as well as chemokine receptors (10) are thought to provide an address code for leukocyte migration to the various sites of inflammation (11,12). Of the numerous adhesion molecules and chemokine receptors several are also used by pathogenic microbes as co-receptors for adhesion and cellular entry. For example, CCR5 and CXCR4 chemokine receptors are the main co-receptors that HIV uses for cellular entry (13). As the use of the host adhesion and chemokine receptors has provided an evolutionary advantage for the microbe, the function of these host receptors is especially interesting.
To assess the address codes to the middle ear mucosa, we analyzed the expression of the chemokine receptors CXCR4 and CCR5 and the lymphocyte homing receptor L-selectin in CD4+ T lymphocytes in middle ear effusion. The expression of these surface markers was then determined in the adenoidal CD4+ T lymphocytes in search for CD4+ T lymphocytes having a potential capacity to home to the middle ear mucosa.
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Methods
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Patients
Tissue samples were obtained from patients operated at the Department of Otorhinolaryngology, Helsinki University Central Hospital. Adenoids were obtained from 13 children suffering from OME and from 12 children with adenoid hyperplasia resulting in obstructive symptoms. Tonsils were obtained from eight children suffering from tonsillar hyperplasia leading to obstructive symptoms. Cervical jugulodigastric lymph nodes were obtained from five patients that were operated on for brachial cleft cysts of the neck. Peripheral blood samples were taken from four individuals of the 13 undergoing adenoidectomy for OME. The study has been approved by the Ethical Review Board of the Department of Otorhinolaryngology, Helsinki University Central Hospital.
Preparation of lymphocytes
Tissue specimens were homogenized using a Medimachine (Dako, Glostrup, Denmark) tissue disaggregation machine to obtain a single-cell suspension. The suspension was then filtered through 50 µm disposable filters (Dako). After washing, the cells were suspended in growth medium RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with 1 mM glutamine (Gibco, Chagrin Falls, OH), antibiotics (penicillin 100 U/ml and streptomycin 100 µg/ml) and 10% human blood group AB serum (Finnish Red Cross Blood Service, Helsinki, Finland) at a cell concentration of 25x106/ml. Peripheral blood lymphocytes were separated with Ficoll.
Middle ear aspirates
Middle ear effusion was collected from children with OME undergoing tympanostomy tube insertion in general anesthesia. The effusion was aspirated through incision in the ear drum. The aspirate was immediately suspended to RPMI 1640 growth medium containing 2 mM EDTA and antibiotics. The specimens were transferred to the laboratory, aliquoted for staining, centrifuged and stained with fluorochrome-conjugated antibodies. Altogether, aspirates from 10 middle ears were collected, stained with antibodies and analyzed. Data from four aspirates that contained the highest number of live lymphocytes is presented. The number of live lymphocytes ranged from 20,000 to 1000 in each analysis. The middle ear cavity in healthy individuals does not contain any effusion and it is filled with air that gets there during swallowing.
Antibodies and flow cytometry
Lymphocytes were stained with fluorochrome-conjugated mAb after which the distribution of different cell types was analyzed using a FACScan flow cytometer with CellQuest software (Becton Dickinson, San Jose, CA). The following fluorochrome-conjugated mAb were used: FITC and PerCPanti-CD3 (clone SK7; Becton Dickinson), FITC and phycoerythrin (PE)anti-CD45RO (clone UCHL1; PharMingen), PEanti-CCR5 (clone 2D7/CCR5; PharMingen), PEanti-CXCR4 (clone 12G5; PharMingen), PE and CyCromeanti-L-selectin (clone DREG-56, PharMingen), PEanti-CD69 (clone FN50, PharMingen) and CyCromeanti-CD4 (clone RPA-T4; PharMingen). CD4+ T lymphocytes in the adenoids, the tonsils, and the lymph node were identified by gating the cells on basis of side scatter and expression of CD4. This cell population expressed CD3 (on the average 97.3% of the cells were positive for CD3, range 9599%). The CD4+, CD3 cell population consisted mainly of cells positive for the dendritic cell maker CD1a.
Purification of CD4+ T lymphocytes
T lymphocyte populations were purified using mAb labeled with magnetic beads. The magnetically labelled cells were separated with LS+ separation columns using MidiMACS Separation unit instrumentation (Miltenyi Biotec, Bergisch Gladbach, Germany).
A three-step purification protocol was used to purify L-selectin and positive CD45RO+, CD4+ T lymphocytes: (i) positive selection of CD4+ cells, (ii) negative selection of CD14+ and CD45RA+ cells, and (iii) purification of L-selectin+ and L-selectin cells with anti-L-selectin antibodies.
Four hundred million adenoidal cells were suspended in 3.2 ml of separation buffer (PBS, pH 7.4, 0.5% BSA, 2 mM EDTA) and 200 µl of CD4 MultiSort MicroBeads (Miltenyi Biotec), and were incubated at 6°C for 15 min. Subsequently, the cells were washed and suspended in 2 ml of ice-cold separation buffer. The cell suspension was eluted through an LS+ separation column, washed, subsequently removed from the magnet and eluted with 5 ml of separation buffer. The magnetic particles were removed from the eluted cells with 100 µl of Release Reagent (Miltenyi Biotec) applied for 10 min in the refrigerator. The cells were washed, suspended in 250 µl of separation buffer and 200 µl of Stop Reagent (Miltenyi Biotec) was added. The CD4+ fraction contained 5080x106 cells. Subsequently, 100 µl of CD45RA MicroBeads (Miltenyi Biotec) and 100 µl of CD14 MicroBeads (Miltenyi Biotec) were added, and the cell suspension was incubated at 6°C for 15 min. The cells were washed, suspended in 1 ml of separation buffer, eluted through the LS+ column, washed with 3 ml of separation buffer and the unbound eluate was recovered. The eluate contained 3050x106 cells. Thereafter 150 µl of PE-conjugated anti-CD62L antibody was added to 15x106 cells in 120 µl of separation buffer. The suspension was incubated at 6°C for 20 min, after which the cells were washed and 30 µl of anti-PE MicroBeads (Miltenyi Biotec) was added for 15 min. After this the cells were washed, suspended in 0.5 ml of separation buffer and eluted through LS+ separation column. The column was washed with 3 ml of separation buffer (negative fraction), removed from the magnet and eluted with 5 ml of separation buffer (positive fraction). The positive and negative fractions contained 57x106 cells.
A two-step purification protocol was used to purify CD4+ T lymphocytes: (i) positive selection of CD4+ cells and (ii) negative selection of CD14+ cells. Ten million adenoidal cells were used for the purification. The purification steps were scaled down according to manufacturers instructions (Miltenyi Biotec).
IFN-
Purified cell preparations were stimulated with phorbol myristate acetate (PMA; Sigma, St Louis, MO) and ionomycin (Calbiochem, La Jolla, CA) in the presence of Brefeldin A (Sigma). The cells were aliquoted into tubes at a concentration of 2x106 /ml in 1 ml of RPMI 1640 (Gibco) supplemented with 36 µM Brefeldin A, 10% FCS, 1 mM glutamine (Gibco) and antibiotics (penicillin 100 U/ml and streptomycin 100 µg/ml; Gibco). Then 40 nM PMA and 1.3 µM ionomycin were added for stimulation. Neither PMA nor ionomycin was added in control tubes. The cell preparations were incubated for 4 h in a 5% CO2/95% humidified air atmosphere at 36°C. The cells were harvested and stained with PerCP-conjugated anti-CD3 antibody (Becton Dickinson; clone SK7). Intracellular staining for IFN-
was done according to the Becton Dickinson FastImmune Cytokine System. Briefly, 2 ml of FACS Lysing solution was added to each tube, incubated 10 min and centrifuged for 5 min. The supernatant was removed and 500 µl of FACS permeabilizing solution was added. The cells were incubated 10 minutes and washed with 3 ml RPMI 1640 and centrifuged. Subsequently the cells were stained with fluorescein-conjugated anti-IFN-
antibody (Becton Dickinson; clone 25723.11) for 30 min, washed and fixed with 1% paraformaldehyde. IFN-
production was measured by flow cytometry in cells gated on the basis of side scatter and CD3 expression. Stock solutions (500-fold of the working dilution) of PMA (12.5 µg/ml, 20.3 µM), ionomycin (0.5 mg/ml, 670 µM) and Brefeldin A (5 mg/ml, 17.8 mM) were prepared in DMSO, aliquoted and stored at 20°C.
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Results
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In middle ear effusion, most CD4+, CD3+ T lymphocytes were positive for CXCR4, CCR5 and the memory phenotype marker CD45RO, but were negative for L-selectin (Fig. 1
). In the adenoids and peripheral blood, subpopulations of CD4+ T lymphocytes could be distinguished by the expression of CD45RO, L-selectin, CXCR4 and CCR5 (Fig. 2
). Adenoidal and peripheral blood naive phenotype (CD4+, CD45RO) cells were homogenous in L-selectin, CXCR4 and CCR5 expression pattern, and resembled each other by expressing L-selectin at a uniformly high level, and by expressing CXCR4, but not CCR5 (Fig. 2
). Adenoidal and peripheral blood memory phenotype (CD4+, CD45RO+) cells were more heterogeneous, and distinct expression patterns of L-selectin, CXCR4 and CCR5 could be observed (Fig. 2
).

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Fig. 1. Expression of CD45RO, L-selectin, CXCR4 and CCR5 in CD4+, CD3+ T lymphocytes in the middle ear effusion. Cells were stained with FITCanti-CD3, CyCromeanti-CD4 and PEIgG2a isotype control antibody (A), PEanti-CD45RO (B), PEanti-L-selectin (C), PEanti-CXCR4 (D), and PEanti-CCR5 (E). T lymphocytes were gated on the basis of side scatter and CD3 expression. The cell surface expression of CD45RO, L-selectin, CXCR4 and CCR5 (y-axis) is plotted against the expression of CD4 (x-axis) in middle ear CD3+ T lymphocytes. The lines present the cut-off values used for the enumeration of positive and negative cells. The percentages of cells in each quadrant are given. The level of staining used as the cut-off to define CCR5bright cells is indicated by the dashed line.
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Fig. 2. Expression of L-selectin, CXCR4 and CCR5 (y-axis) as plotted against the expression of CD45RO (x-axis) in adenoidal and peripheral blood CD4+ lymphocytes. Adenoidal and peripheral blood cells were stained with CyCromeanti-CD4, FITCanti-CD45RO and PEIgG2a isotype control antibody (A and B), PEanti-L-selectin (C and D), PEanti-CXCR4 (E and F), and PEanti-CCR5 (G and H). CD4+ lymphocytes were gated on the basis of side scatter and CD4 expression. The lines indicate the level of staining used as cut-offs to enumerate CD4+ lymphocyte subsets. The percentages of cells in each quadrant are given. The level of staining used as the cut-off to define CCR5bright cells is indicated by the dashed line.
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Memory phenotype (CD4+, CD45RO+) cells both in the adenoids and peripheral blood included both L-selectin+ and L-selectin cells. In peripheral blood most memory phenotype cells were L-selectin+ but in the adenoids a considerable proportion of memory phenotype cells were L-selectin (Fig. 2
).
The level of CXCR4 expression in memory phenotype (CD4+, CD45RO+) cells was also variable in the adenoids and peripheral blood. In peripheral blood most memory phenotype cells were CXCR4 or expressed CXCR4 at a low level, but in the adenoids most CD4+, CD45RO+ cells were CXCR4+ and only few were negative for CXCR4. Moreover, the level of CXCR4 expression was high in adenoidal memory phenotype (CD4+, CD45RO+) cells, even higher than in adenoidal naive phenotype (CD4+, CD45RO) cells (Fig. 2
).
A minority of memory phenotype (CD4+, CD45RO+) cells both in the adenoids and in peripheral blood expressed CCR5 (Fig. 2
). The main difference between the adenoids and peripheral blood was in that a small subpopulation of adenoidal memory phenotype cells expressed CCR5 at a high level but this phenotype was considerably less frequent in blood. The CCR5bright cells could also be observed in the middle ear effusion (Fig. 1
).
The surface phenotype of adenoidal memory phenotype (CD4+, CD45RO+) cells thus resembled the surface phenotype of CD4+ cells in middle ear effusion, in that the former included CD4+ memory phenotype (CD45RO+) cells that were L-selectin, CXCR4+ or CCR5bright. The frequencies of L-selectin+, CXCR4+ and CCR5bright cells in adenoidal and peripheral blood memory phenotype (CD45RO+, CD4+) cells and in CD4+ T lymphocytes in the middle ear effusion are presented in Fig. 3
.

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Fig. 3. Frequencies of L-selectin+, CXCR4+ and CCR5bright cells in CD45RO+, CD4+ memory phenotype cells in the adenoids and peripheral blood and in CD4+, CD3+ T lymphocytes in the middle ear effusion. Adenoidal and peripheral blood cells were stained and enumerated as described in Fig. 2 . The percentages of L-selectin+ cells (A), CXCR4+ cells (B) and CCR5bright cells (C) of all CD4+, CD45RO+ cells are illustrated in the adenoids and peripheral blood of four children. Dots presenting the percentages of cells in the adenoids and peripheral blood of the same individual are connected with lines. Horizontal lines present the mean percentages of the four children. The mean percentage of L-selectin+ cells of all CD4+, CD45RO+ cells was higher in peripheral blood as compared to the adenoids (A, P = 0.022, Student's t-test of paired values). The mean percentage of CXCR4+ cells of all CD4+, CD45RO+ cells was lower in peripheral blood as compared to the adenoids (B, P = 0.006). The mean percentage of CCR5bright cells of all CD4+, CD45RO+ cells was lower in peripheral blood as compared to the adenoids (C, P = 0.038). The percentages of L-selectin+ cells, CXCR4+ cells and CCR5bright cells of all CD4+, CD3+ T cells in the middle ear effusion of four different children are illustrated in (D), (E) and (F) respectively. Horizontal lines present the mean percentages. The frequencies of CD45RO+ cells in CD3+, CD4+ cells in four middle ear effusions were 98, 96, 90 and 85%.
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To further characterize the adenoidal CD4+, CD45RO+ lymphocytes, adenoidal CD4+ cells were purified with magnetic beads and triple stained either with CD45RO, L-selectin, and CXCR4 antibodies or with CD45RO, L-selectin and CCR5 antibodies (Fig. 4
). Most L-selectin and L-selectin+ cells expressed CXCR4. However, the L-selectin and L-selectin+ populations differed from each other in that the L-selectin population contained approximately twice as much CCR5bright cells as the L-selectin+ population (Fig. 4
).

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Fig. 4. Expression of CXCR4 and CCR5 as plotted against the expression of L-selectin in adenoidal CD4+, CD45RO+ T lymphocytes. Adenoidal CD4+ T lymphocytes were purified as described in Methods. The purification resulted in a cell population that was >98% CD3+ and <1% CD8+. The purified cells were stained simultaneously with FITCanti-CD45RO, PEanti-CXCR4 and with CyCromeanti-L-selectin (A) or with FITCanti-CD45RO, PEanti-CCR5 and with CyCromeanti-L-selectin (B). The stained cells were gated by the expression of CD45RO and side scatter. The expression of L-selectin was plotted against the expression of CXCR4 (A) and CCR5 (B) in the gated CD45RO+ cells. The lines indicate the cut-off values used for the enumeration of the cells. The percentages of CXCR4+ (A) and CCR5high cells (frame B) in L-selectin high and in L-selectin or low cells are given. The CCR5bright cells were more frequent in L-selectin or low cells as compared with L-selectinhigh cells. Similar results were obtained in two other experiments.
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To characterize the L-selectin and L-selectin+ CD4+, CD45RO+ cell populations even further, they were purified using magnetic beads and their IFN-
production was measured by flow cytometry. However, obvious differences in IFN-
production were not observed (Fig. 5
).
To test if the L-selectin memory phenotype (CD4+, CD45RO+) population was characteristic for the adenoids, the frequency of L-selectin+ (CD4+, CD45RO+) cells in tonsils and in cervical jugulodigastic lymph nodes were studied (Fig. 6
). It was found that the frequency of L-selectin+, CD4+, CD45RO+ cells in the lymph node was high as compared to the adenoids or to the tonsils. It was also found that the disease history of the child was associated with the expression of L-selectin in CD4+, CD45RO+ cells of the adenoids. Children who had been operated on because of OME had a higher proportion of adenoidal L-selectin memory phenotype cells than children operated on because of obstructive symptoms (Fig 6
).

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Fig. 6. Percentages of L-selectin+ cells of all CD4+, CD45RO+ cells in the adenoids, tonsils and cervical lymph node. One dot represents the value of one tissue specimen. The values for the adenoids obtained from children suffering from OME (Adenoids, otitis) and the values for the adenoids obtained from children that were operated because of enlargement of the adenoids resulting in obstructive symptoms (Adenoids, obstruction) are plotted in separate columns. The mean percentages in each column are indicated by bars and are given in parenthesis. The mean percentage in the `Adenoids, otitis' group was lower than in the `Adenoids, obstruction' group (P = 0.009, Student's t-test of independent samples). The age distributions of these two groups of patients were close to each other. The mean age in the `Adenoids, otitis' group was 7.1 years (range 4.711.8, n = 9) and the mean age in the `Adenoids, obstruction' group was 7.0 years (range 5.011.3 years, n = 12). Lymph nodes had on the average higher concentrations of L-selectin+ CD4+, CD45RO+ cells than the tonsils (P = 0.023) and the adenoids (P = 0.018, children with obstructive symptoms, P < 0.001 children with OME).
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As cellular activation is sometimes associated with down-regulation of L-selectin, we studied the expression of the early activation antigen CD69 in T lymphocytes of the middle ear effusion. Most middle ear T lymphocytes, which were mainly L-selectin, were also negative for CD69 (Fig. 7
). Adenoidal memory phenotype (CD4+, CD45RO+) T lymphocytes, which included many L-selectin cells, were mostly positive for CD69. Lymph node memory phenotype CD4+, CD45RO+ T lymphocytes, which were different to their adenoidal counterparts in that they were mainly L-selectin+, also expressed CD69. Naive phenotype (CD4+, CD45RO) adenoidal T lymphocytes were positive for L-selectin and negative for the early activation antigen CD69 (data not shown).

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Fig. 7. Expression of L-selectin and CD69 in CD4+, CD45RO+ cells in the middle ear effusion, in the adenoids and in the lymph node. Cells were stained with FITCanti-CD45RO, PEanti-L-selectin and CyCromeanti-CD4 (AC) and with FITCanti-CD45RO, PEanti-CD69 and CyCromeanti-CD4 (DF). Cells were gated on the basis of CD4 expression and low side scatter (AC) and the expression of L-selectin (y-axis) was plotted against the expression of CD45RO (x-axis). The percentages of L-selectin+ cells of all CD4+, CD45RO+ cells are given in (A)(C). In (D)(F) cells were gated on the basis of CD4 and CD45RO positivity and low side scatter. The expression of CD69 (x-axis) was plotted against the cell count (y-axis). The percentages of CD69+ cells of all CD4+, CD45RO+ cells are given in (D)(F) (middle ear effusion, adenoids and cervical lymph node respectively). Staining with PEIgG2a isotype control antibody gave no positive cells when the value indicated by the lines in (D)(F) was used as the cut-off.
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Discussion
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Middle ear effusion was enriched in CD4+ T lymphocytes of a distinct phenotype. These CD4+ T lymphocytes expressed the memory phenotype marker CD45RO, and high levels of the chemokine receptors CXCR4 and CCR5, but were negative for the lymphocyte homing receptor L-selectin. This phenotype was rare in peripheral blood but was found relatively frequently in the adenoids. Interestingly, the L-selectin, CD4+, CD45RO+ phenotype found at a high concentration in the adenoids was relatively rare in the jugulodigastric lymph nodes (of the neck) in patients operated for infected brachial cleft cysts. These results suggest that the microenvironments in the middle ear effusion and in the adenoids are unique in sustaining high-level expression of CXCR4 and CCR5, and in down-regulation of L-selectin in CD4+, CD45RO+ T lymphocytes.
The mechanism of L-selectin down-regulation in adenoidal or middle ear lymphocytes is not known. L-selectin is essential in lymphocyte migration to peripheral lymph nodes and to cutaneous sites of inflammation as shown in transgenic mice (1417). Also human tissue lymphocytes in cutaneous inflammation have been found to express L-selectin (18). L-selectin is shed from the cell surface by an endoproteolytic mechanism (19,20) and in vivo it is thought to occur at the endothelial surface before lymphocyte extravasation (19). As L-selectin shedding is sometimes associated with stimuli leading to lymphocyte proliferation (21) we measured the expression of the early activation antigen CD69 in lymphocytes of the middle ear effusion. Most middle ear CD4+ T cells were negative for both CD69 and L-selectin. Adenoidal and lymph node CD4+, CD45RO+ T lymphocytes, which differed in their L-selection expression profiles, both expressed CD69. This suggests that in vivo proliferation as such is not necessarily associated with L-selectin down-regulation. The mechanism of L-selectin down-regulation in the adenoids may resemble the mechanism that occurs in the intestinal lamina propria lymphocytes from Rhesus monkeys which do not express L-selectin or L-selectin mRNA (22).
In addition to the middle ear, CD4+ T lymphocytes in other mucosal sites have been reported to be L-selectin. As mentioned above, intestinal lamina propria lymphocytes lack L-selectin expression (22). Also human brochoalveolar lavage fluid (18) and mouse gastric mucosa (23) lymphocytes have been reported to be L-selectin. It is possible that L-selectin is not required for lymphocyte migration into mucosal sites. Experiments with transgenic mice have revealed that L-selectin expression is not a necessity for lymphocyte migration to intestinal Peyer's patches (14). In mice challenged with peritoneal superantigen, antibodies to CD44 but not to L-selectin inhibit T lymphocyte infiltration to peritoneal mucosa (24).
Previously, the lack of L-selectin expression in human peripheral blood has been associated with an increased ability to produce IFN-
(25). CCR5-expressing cells, which have been reported to be enriched in cells producing Th1 type of cytokines such as IFN-
(26), have also been reported to be enriched in L-selectin, CD45RO+, CD4+ cells (27). The findings above suggested that adenoidal L-selectin cells may have increased production of IFN-
. However, we failed to detect significant differences in IFN-
production between purified L-selectin and L-selectin+, CD45RO+, CD4+ populations. It was also found that the overall production of IFN-
in adenoidal CD3+ cells was low when compared to peripheral blood CD3+ cells (data not shown). It is possible that continuous in vivo activation of adenoidal T cells makes them unresponsive to stimulation. Continuous in vivo activation and unresponsiveness to stimulation has also been observed to occur in human lamina propria T lymphocytes (28). While functional differences between cell subpopulations may exist, it appears that the ability of adenoidal CD4+, CD45RO+ T lymphocytes to produce IFN-
is not strongly associated with the expression of L-selectin.
The expression of the chemokine receptors CXCR4 and CCR5 in the CD4+ cells of middle ear effusion implies that CXCR4 and CCR5 may be important in lymphocyte recruitment to the middle ear mucosa. CXCR4 and CCR5 may be important in lymphocyte homing to certain other mucosal sites in addition to the middle ear such as to intestinal lamina propria which has been found to contain CXCR4 and CCR5+ T lymphocytes (29). CCR5-expressing T cells have also been found in synovial fluid (30) which also represents a mucosal site. On the other hand, it is possible that CXCR4 and CCR5 are not important in extravasation in the middle ear per se but are up-regulated after extravasation. This is feasible as the surrounding microenvironment may modulate the expression of chemokine receptors (3133).
The high expression of both CXCR4 and CCR5 in adenoidal and middle ear CD45RO+, CD4+ T lymphocytes was unexpected as they have been reported to be differentially expressed and regulated on human T lymphocytes (31). In human circulating CD4+ T lymphocytes CCR5 expression is found predominantly in memory phenotype CD45RO+ cells but CXCR4 is expressed predominantly in naive phenotype CD45RO, CD4+ T cells (27,31). Such a expression pattern of CXCR4 and CCR5 was also observed in CD4+ T lymphocytes in the peripheral blood of our patients.
As adenoidal memory phenotype CD4+, CD45RO+ T cells expressed CXCR4, CCR5 and the early activation antigen CD69, they may be permissive for infection by HIV which preferentially infects activated memory phenotype CD4+ cells (34) and which use CXCR4 (X4 strains) and CCR5 (R5 strains) as co-receptors (13,35). The putative permissiveness of adenoidal CD4+ T cells to HIV infection may be related to the frequent presence of HIV in the adenoids of asymptomatic HIV patients (36).
That the major type of the middle ear CD4+ T cells (CD45RO+, L-selectin, CXCR4+, CCR5+) was also found in the adenoids raises the possibility that in the adenoids this CD45RO+, L-selectin, CXCR4+, CCR5+ CD4+ T cell population may include cells that eventually home to the middle ear mucosa. Adenoidectomy in children with OME may thus decrease the generation of lymphocytes capable of homing to the middle ear and thereby reduce the inflammatory reaction in the middle ear. It is possible that in children with persistent otitis media the generation of adenoidal lymphocytes capable of homing to the middle ear may be increased as these children had an increased proportion of adenoidal L-selectin memory phenotype CD4+ cells. It should be noted, however, that adenoidectomy may have multiple effects on the local immune response in the nasopharynx and that a reduced lymphocyte homing to the middle ear mucosa may only partly explain the effects of adenoidectomy.
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Acknowledgments
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This work received financial support from the Paulo Foundation, Helsinki, Finland. We thank Ms Leena Juvonen, Ms Kirsi Ijäs and Ms Tuija Huhtala for excellent technical assistance. We are particularly grateful for Manuela Mengozzi and Klaus Hedman for careful reading of the manuscript and constructive discussions.
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Abbreviations
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OME otitis media with effusion |
PE phycoerythrin |
PMA phorbol myristate acetate |
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Notes
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Transmitting editor: D. Tarlinton
Received 3 March 2000,
accepted 10 May 2000.
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References
|
---|
-
Maw, A. R. 1983. Chonic otitis media with effusion (glue ear) and adenotonsillectomy: a prospective randomized controlled study. Br. Med. J. 287:1586.[ISI][Medline]
-
Gates, G. A., Avery, C. A., Cooper, J. C. and Prihoda, T. J. 1987. Effectiveness of adenoidectomy and tympanostomy tubes in the treatment of chonic otitis media with effusion. N. Engl. J. Med. 317:1444.[Abstract]
-
Sade, J. and Luntz, M. 1991. Adenoidectomy in otitis media: a review. Ann. Otol. Rhinol. Laryngol. 100:226.[ISI][Medline]
-
Gates, G. A., Muntz, H. R. and Gaylis, B. 1992. Adenoidectomy and otitis media. Ann. Otol. Rhinol. Laryngol. Suppl. 155:24.[Medline]
-
Fearon, M., Bannatyne, R. M., Fearon, B. W., Turner, A. and Cheung, R. 1992. Differential bacteriology in adenoid disease. J. Otolaryngol. 21:434.[ISI][Medline]
-
Forsgren, J., Samuelson, A., Lindberg, A. and Rynnel, D. B. 1993. Quantitative bacterial culture from adenoid lymphatic tissue with special reference to Haemophilus influenzae age-associated changes. Acta Otolaryngol. 113:668.[ISI][Medline]
-
Linder, T. E., Marder, H. P. and Munzinger, J. 1997. Role of adenoids in the pathogenesis of otitis media: a bacteriologic and immunohistochemical analysis. Ann. Otol. Rhinol. Laryngol. 106:619.[ISI][Medline]
-
Butcher, E. C. and Picker, L. J. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
-
Springer, T. A. 1990. Adhesion receptors of the immune system. Nature 346:425.[ISI][Medline]
-
Baggiolini, M., Dewald, B. and Moser, B. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675.[ISI][Medline]
-
Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033.[ISI][Medline]
-
Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[ISI][Medline]
-
Berger, E. A., Murphy, P. M. and Farber, J. M. 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657.[ISI][Medline]
-
Arbones, M. L., Ord, D. C., Ley, K., Ratech, H., Maynard-Curry, C., Otten, G., Capon, D. J. and Tedder, T. F. 1994. Lymphocyte homing and leucocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity 1:247.[ISI][Medline]
-
Steeber, D. A., Green, N. E., Sato, S. and Tedder, T. F. 1996. Lyphocyte migration in L-selectin-deficient mice. Altered subset migration and aging of the immune system. J. Immunol. 157:1096.[Abstract]
-
Tedder, T. F., Steeber, D. A. and Pizcueta, P. 1995. L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. J. Exp. Med. 181:2259.[Abstract]
-
Tang, M. L., Hale, L. P., Steeber, D. A. and Tedder, T. F. 1997. L-selectin is involved in lymphocyte migration to sites of inflammation in the skin: delayed rejection of allografts in L-selectin-deficient mice. J. Immunol. 158:5191.[Abstract]
-
Picker, L. J., Martin, R. J., Trumble, A., Newman, L. S., Collins, P. A., Bergstresser, P. R. and Leung, D. Y. 1994. Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites. Eur. J. Immunol. 24:1269.[ISI][Medline]
-
Chen, A., Engel, P. and Tedder, T. F. 1995. Structural requirements regulate endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J. Exp. Med. 182:519.[Abstract]
-
Feehan, C., Darlak, K., Kahn, J., Walcheck, B., Spatola, A. F. and Kishimoto, T. K. 1996. Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor. Identification with an L-selectinalkaline phosphatase reporter. J. Biol. Chem. 271:7019.[Abstract/Free Full Text]
-
Frey, M., Appenheimer, M. M. and Evans, S. S. 1997. Tyrosine kinase-dependent regulation of L-selectin expression through the Leu-13 signal transduction molecule: evidence for a protein kinase C-independent mechanism of L-selectin shedding. J. Immunol. 158:5424.[Abstract]
-
Berg, M., Murakawa, Y., Camerini, D. and James, S. P. 1991. Lamina propria lymphocytes are derived from circulating cells that lack the leu-8 lymph node homing receptor. Gastroenterology 101:90.[ISI][Medline]
-
Katakai, T., Mori, K. J., Masuda, T. and Shimizu, A. 1998. Differential localization of Th1 and Th2 cells in autoimmune gastritis. Int. Immunol. 10:1325.[Abstract]
-
De Grendele, H. C., Estess, P. and Siegelmann, M. H. 1997. Requirement of CD44 in activated T cell extravasation into an inflammatory site. Science 278:672.[Abstract/Free Full Text]
-
Kanegane, H., Kasahara, Y., Niida, Y., Yachie, A., Sughii, S., Takatsu, K., Taniguchi, N. and Miyawaki, T. 1996. Expression of L-selectin (CD62L) discriminates Th1- and Th2-like cytokine-producing memory CD4+ T cells. Immunology 87:186.[ISI][Medline]
-
Bonecchi, R., Bianchi, G., Bordignon, P. P., D'Ambrosio, D., Lang, R., Borsatti, A., Sozzani, S., Allavena, P., Gray, P. A., Mantovani, A. and Sinigaglia, F. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129.[Abstract/Free Full Text]
-
Lee, B., Sharron, M., Montaner, L. J., Weissman, D. and Doms, R. W. 1999. Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. Proc. Natl Acad. Sci. USA 96:5215.[Abstract/Free Full Text]
-
De Maria, R., Fais, S., Silvestri, M., Frati, L., Pallone, F., Santoni, A. and Testi, R. 1993. Continuous in vivo activation and transient hyporesponsiveness to TcR/CD3 triggering of human gut lamina propria lymphocytes. Eur. J. Immunol. 23:3104.[ISI][Medline]
-
Lapenta, C., Boirivant, M., Marini, M., Santini, S. M., Logozzi, M., Viora, M., Belardelli, F. and Fais, S. 1999. Human intestinal lamina propria lymphocytes are naturally permissive to HIV-1 infection. Eur. J. Immunol. 29:1202.[ISI][Medline]
-
Suzuki, N., Nakajima, A., Yoshino, S., Matsushima, K., Yagita, H. and Okumura, K. 1999. Selective accumulation of CCR5+ T lymphocytes into inflamed joints of rheumatoid arthritis. Int. Immunol. 11:553.[Abstract/Free Full Text]
-
Bleul, C. C., Wu, L., Hoxie, J. A., Springer, T. A. and Mackay, C. R. 1997. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc. Natl Acad. Sci. USA 94:1925.[Abstract/Free Full Text]
-
Sallusto, F., Kremmer, E., Palermo, B., Hoy, A., Ponath, P., Qin, S., Forster, R., Lipp, M. and Lanzavecchia, A. 1999. Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells. Eur. J. Immunol. 29:2037.[ISI][Medline]
-
Cole, S. W., Jamieson, B. D. and Zack, J. A. 1999. cAMP up-regulates cell surface expression of lymphocyte CXCR4: implications for chemotaxis and HIV-1 infection. J. Immunol. 162:1392.[Abstract/Free Full Text]
-
Roederer, M., Raju, P. A., Mitra, D. K., Herzenberg, L. A. and Herzenberg, L. A. 1997. HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. J. Clin. Invest. 99:1555.[Abstract/Free Full Text]
-
D'Souza, M. P. and Harden, V. A. 1996. Chemokines and HIV-1 second receptors. Confluence of two fields generates optimism in AIDS research. Nat. Med. 2:1293.[ISI][Medline]
-
Frankel, S. S., Wenig, B. M., Burke, A. P., Mannan, P., Thompson, L. D., Abbondanzo, S. L., Nelson, A. M., Pope, M. and Steinman, R. M. 1996. Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid. Science 272:115.[Abstract]