Ann Otol Rhinol Lary"6f'llOl:l992










Adenoidal lymphocytes obtained from 43 subjects with serum antibodies to respiratory syncytial virus (RSV) were established in culture in vitro and analyzed for immunoglobulin (lg) and RSV -specific antibody synthesis. Spontaneous synthesis ofIg was consistently observed in culture supernatants. The ratios ofIgA to IgG and IgM to IgG in adenoidal lymphocyte culture supernatant were higher than in serum. In cell cultures stimulated with RSV or pokeweed mitogen, RSV antibody activity was detected in 25 of 43 (58.1 %) for IgG, 5 of 43 (11.6%) for IgA, and 4 of 43 (9.3%) for IgM. Also, RSV-specific IgG was detected in some supernatants from unstimulated cultures. In seven cases the cultures of autologous tonsillar lymphocytes were also investigated. Autologous organs exhibited similar polyclonal Ig production and RSV -specific antibody synthesis. These observations demonstrate that both adenoids and palatine tonsils are continuously engaged in synthesis of local antibodies to viral pathogens available to the nasopharynx and respiratory mucosa. KEY WORDS -

adenoidal lymphocytes, antibody production, respiratory syncytial virus.

treatment of eustachian tube obstruction and otitis media in childhood (review by Sade and Luntz!"), The present investigations were undertaken to evaluate the relative contribution of adenoid tissue lymphocytes in the production of polyclonal Ig and in the synthesis and release of virus-specific antibody activity in the naso-oropharynx by employing in vitro synthesis of RSV antibody in human adenoidal cell cultures.


The pharyngeal tonsil (adenoid), together with the palatine tonsils and other lymphoid tissues, forms Waldeyer's ring and is thought to play an important role in immunity, especially to the antigens available in the nasal or oral cavities. 1 Much of the evidence suggesting this role is derived from immunohistochemical or proliferation studies.e? However, there is only limited quantitative information available on synthesis and release of polyclonal immunoglobulin (lg) or specific antibodies by adenoids. 4,IO,11


Study Subjects. Adenoids were obtained from 43 patients (23 boys and 20 girls) suffering from adenoidal hypertrophy associated with snoring, otitis media, or chronic hypoxia and known to have serum antibodies against RSV. The subjects ranged from 2 to 16 years in age. Adenoidectomy was carried out during a community outbreak ofRSV infection (November to April) in 29 patients, during non-RSV season (August to October) in 10 patients, and during other times of the year in the remaining 4 subjects. Tonsils were also obtained at the same time from 7 patients (5 boys and 2 girls) suffering from chronic tonsillitis or upper respiratory tract obstruction. These

Respiratory syncytial virus (RSV) is one of the most important pathogens that cause severe respiratory diseases in infants. 12 Recent investigations have suggested that lymphocytes from palatine tonsils synthesize specific antibodies to many respiratory pathogens, including RSV, when stimulated in vitro and possibly in vivo. 13 Such antibody response may participate in local immunity to a variety of pathogens of the upper airway, middle ear, and paranasal sinuses. Recently, the use of adenoidectomy alone has been suggested as an effective approach to prevention and

From the Departments of Pediatrics and Microbiology, State University of New York at Buffalo, and the Division of Infectious Diseases and MicrobiologyLaboratories,Children's Hospitalof Buffalo,Buffalo,New York(Soh, Nadal,Schlapfer,Bernstein),and theDepartmentof Pediatrics, UniversityofTexas MedicalBranch,Galveston,Texas (Ogra). Dr Soh is currentlyat the Departmentof Otorhinolaryngology,Facultyof Medicine, KyushuUniversity,Fukuoka,Japan. Supported in part by a grant from the NationalInstitutes of HealthlNationalInstitute of Allergy and Infectious Diseases (AI-15939). REPRINTS - Pearay L. Ogra, MD, Dept of Pediatrics, CD-51, University of Texas Medical Branch at Galveston Children's Hospital, Galveston, TX 77555-0351.


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Soh et al, Adenoidal Lymphocytes

subjects ranged from 2 to 8 years of age. Virus. HEp-2 cell culture monolayers were maintained in minimal essential medium (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco). The long strain ofRSV was grown in HEp-2 cell cultures to a stock titer of 106plaque-forming units (PFU) per milliliter. Antigen for the enzyme-linked immunosorbent assay (ELISA) was prepared by partial purification of RSV.I s In brief, RSV was separated from the culture fluid by polyethylene glycol precipitation, suspended in 20% sucrose-NTE buffer (0.15 mollL NaC1; 0.05 mo1/LTris-HC1; 1mo1/Lethylenediaminetetraacetic acid; pH 7.5), and then centrifuged through a 30% sucrose-NTE gradient. The pellets were resuspended in 20% sucrose-NTE buffer. Uninfected HEp-2 cells were processed similarly and used as control antigen. Adenoidal or Tonsillar Tissue Lymphocytes. After removal, the adenoids or tonsils were immersed briefly in 95% ethanol and washed in Hanks' balanced salt solution supplemented with 100 U/mL of penicillin, 100 ~g/mL of streptomycin, 100 ~glmL of gentamicin, and 1 ~g/mL of amphotericin B to prevent bacterial contamination. The tissues were minced and passed through a wire mesh. The resulting cell suspension was centrifuged over a Ficoll-Hypaque gradient to separate mononuclear cells from polymorphonuclear cells, erythrocytes, and stromal cells. Separated mononuclear cells (>95% lymphocytes with a viability of 99%) are referred to hereinafter as adenoidal lymphocytes (ALs) or tonsillar lymphocytes (TLs). The AL or TL preparations were partially clarified of cytophilic Ig by incubation for 1 hour at 37°C in RPMI 1640 supplemented with looU/mLofpenicillin, 100 ~g/mL of streptomycin, and 5% heat-inactivated FCS. Cells prepared in this manner were cultured in RPMI 1640 medium supplemented with 50 U/mL of penicillin, 50 ug/ml, of streptomycin, 4 mmol/L of glutamine (Gibco), 5 mmol/L of HEPES buffer, and 10% heat-inactivated FCS (10 million cells per 2 mL) in 24-well tissue culture plates (Linbro, McLean, Va). Triplicate cultures were incubated with live RSV at concentrations ranging from 10,000 (lQ4)to 0.000001 (l0-6)PFU/mLoflivevirus,50~g/ mL pokeweed mitogen (PWM), or medium alone. All cultures were incubated at 37°C in a humid atmosphere supplemented with 5% carbon dioxide in air for 12 days. Detection of Immunoglobulin Synthesis and Release. Immunoglobulin levels in culture supernatants were determined by employing a typical capture ELISA.t 6 Polyclonal rabbit anti human y, n, or ~


chain-specific antisera (Dakopatts, Copenhagen, Denmark) were coated as capture antibodies onto 96well round bottom microtiter plates (Costar, Cambridge, Mass) in 60 ~L of carbonate buffer pH 9.6 and left overnight at 4°C. The optimal dilutions of the antisera were previously determined by checkerboard titration. After washing with phosphate-buffered saline (PBS) plus Tween 20 (0.05%; Sigma, St Louis, Mo; PBS-Tween), serial dilutions of culture supernatants in a final volume of60 ul, per well were added for 1hour at 37°C. Following repeated washings with PBS-Tween, 60 ~ per well of the corresponding polyclonal rabbit anti human heavy chain-specific horseradish peroxidase-conjugated antisera (1:6,000; Dakopatts) were incubated at 37°C for 1 hour. After the final washing step, the enzyme activity bound was detected by using o-phenylenediamine substrate. The color reaction was stopped with 5N H2SO4, and the absorbance at 492 nm was determined by an automated Multiscan ELISA reader (Titertek, Multiscan MCC, Flow Labs, Rockville, Md). All samples were run in duplicate, and the mean of absorption was calculated. The Ig concentrations were calculated by interpolation of the standard curve constructed with serial dilutions of known concentrations of pure human Igs (Dakopatts) included in each plate. The sensitivity ofthe ELISAs was 2 to 3 ng/mL for IgG, 2.8 ng/mL for IgA, and 2.5 ng/mL for IgM. Detection ofAnti-RSVAntibody. The titers of antiRSV IgG, IgA, and IgM were determined by ELISA. 13 Briefly, the wells of the microtiter plates were coated with 100 ~L of partially purified RSV diluted in carbonate buffer (pH 9.6; 2.5 ~g of protein per milliliter) overnight at 4°C. Control wells were coated with partially purified uninfected HEp-2 cells. The wells were washed with PBS-Tween and blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at 37°C. Following three washings with PBS-Tween, the wells were filled with 100 ul, of serial dilutions (starting at 1:4) of culture supernatants in PBS-Tween containing 0.1% BSA and incubated for 12 hours at 4°C. Next, the plates were washed and incubated for 2 hours at 37°C with 100 ~ per well of horseradish peroxidase-conjugated polyclonal rabbit anti human heavy chain-specific antisera. After a final washing step, the color reaction was developed with o-phenylenediamine and stopped after 30 minutes with 5N H2SO4. The end titer of antibody specific for RSV was determined on the basis of the highest sample dilution giving, at 492 om in RSV-coated wells, a twofold higher optical density than in wells coated with the control antigen (cutoff level, optical density of 0.1). All samples were run in duplicate. Serial dilutions of a standard serum with known antibody titer to RSV were also included in

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Soh et al, Adenoidal Lymphocytes


2-6 7-13


IgO (!J.g/mL)

IgA (!J.g/mL)

IgM (!J.g/mL)

11 13

3.88:t 1.89 4.81 :t 1.60

1.74:t 0049 1045 :t 0.28

lAO:t 0.44 2.56:t 0.74

*Immunoglobulins in 12-day culture supernatant of adenoidallymphocytes (5 million cells per milliliter) were determined by EUSA. Values are means :I: SE.

each plate.

Data Analysis. Student's paired t test, Wilcoxon's signed-rank test, and a test were used for statistical analysis. Data are expressed as mean ± SE unless stated otherwise, and values below .05 were regarded as significant.!?


RESULTS Polyclonal Immunoglobulin Production. The spontaneous polyclonal Ig production and release were tested in the lymphocyte cultures of 24 subjects (see Table). Peak levels were found in the 12-day culture supernatants. As shown in the Table, IgG was the predominant isotype, with concentrations over two to three times higher than concentrations of IgA and IgM, respectively. The values for IgA and IgM were similar (see Table). The ALs from children 2 to 6 years of age tended to produce quantitatively lower amounts of IgG than the ALs of older subjects (see Table). The influence of antigenic stimulus on polyc1onal Ig production by ALs is presented in Fig 1. The highest Ig concentrations were measured in supernatants from cultures containing RSV in low concentrations (10-4 or 10-2 PFU/mL), HEp-2, and control

media. Cultures containing higher concentrations of live RSV (100, 102, or 1()4 PFU/mL, respectively), minute amounts of the virus (10-6 PFU/mL), or PWM, respectively, resulted in significantly lower Ig levels (p < .05).

Synthesis ofRSV-Specific Antibodies. The titers of RSV -specific antibodies for the isotypes G, A, and M in AL culture supernatants are presented in Fig 2. Specific IgG was detected in 25 of43 samples(58.1 %), IgA in 5 (11.6%), and IgM in 4 (9.3%; Fig 2). Four of the five samples with specific IgA contained specific IgG, and all samples positive for specific IgM contained specific IgG. The positive titers of specific antibody ranged from

Immunologic response of adenoidal lymphocytes to respiratory syncytial virus.

Adenoidal lymphocytes obtained from 43 subjects with serum antibodies to respiratory syncytial virus (RSV) were established in culture in vitro and an...
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