Journal of Clinical Laboratory Analysis 10:149-154 (1996)

Detection of Salivary and Seric IgG and IgA Antipooled Pigeon Sera Activities in Patients With Pigeon Breeder’s Disease

Felipe Mendoza,’32 Matilde Baltazares,’ Alejandra Ramirez,’ Raul Sansores,’ Arnulfo Nava,’13 Jose Luis Baiiale~:‘’~ and Moises Selman’

71nstituto Nacional de Enfermedades Respiratorias, 2Universidad Autonoma Metropolitana-Unidad Xochimilco, and 31nstituto Nacional de Cardiologia, Mexico D. F:

Pigeon breeder’s disease (PBD) is an in- terstitial lung disease induced by exposure to pigeon antigens. Search of antipigeon antigen antibodies (APSA) in serum or bronchoalveolar lavage is generally used for auxiliary diagnostic purposes. However, APSAcan be present in a number of exposed but asymptomatic individuals as well as in patients with other interstitial lung diseases who live in areas where keeping pigeons is a common domestic habit. In this study, sa- liva was evaluated as an alternative means to serum forAPSA detection by ELISA using pooled pigeon sera as antigen. Serum and saliva samples obtained from 17 patients with PBD, 14 with idiopathic pulmonary fi- brosis (IPF), 19 asymptomatic relatives (AR) exposed to pigeon antigens, and 27 clinical healthy voluntary subjects (CHVS) were tested for IgG and IgA APSA. Our results

showed that both fluids obtained from PBD patients exhibited a significantly higher spe- cific IgG antibody activity compared to the other groups. Serum optical density (O.D.) values for PBD were 1 .I 87 + 0.738 vs. 0.024 *0.033,0.255*0.471, and0.204c0.346for CHVS, AR and IPF, respectively ( P < 0.05). Salivary O.D. for PBD were 0.801 * 0.447 vs 0.010~0.011,0.104~0.151,and0.22~0.447 (P< 0.05). In contrast, serum specific IgA did not discriminate between PBD and IPF pa- tients. In addition, although the PBD group exhibited the highest values of IgA salivary APSA, high levels were also observed in saliva specimens from CHVS, a group of normal individuals who deny pigeon expo- sure. These findings suggest that measure- ment of IgG salivary APSA can play a role in the evaluation process of patients with pi- geon breeder’s disease. 0 1996 Wiley-Liss, Inc.

Key words: hypersensitivity pneumonitis, salivary antibodies, allergic alveolitis, pigeon breeder’s disease, enzymatic immunoassay

INTRODUCTION

Hypersensitivity pneumonitis (HP), or extrinsic allergic alveolitis, is a diffuse inflammation of the lung parenchyma caused by repeated exposure to organic environmental sub- stances with a wide antigenic spectrum (1). One of the most frequent sources of antigens derives from birds, mainly pi- geons, which produce so-called pigeon breeder’s disease (PBD) (2). Gnfortunately, there is no single pathognomic fea- ture for the diagnosis of PBD, especially when the patient presents an acute progressive or a chronic form of the disease (3). Clinical, radiographic, and functional abnormalities are similar to many other interstitial lung diseases, and specific serum-precipitating antibodies might be present in a number of exposed but asymptomatic individuals. Therefore, to ob- tain the precise diagnosis, clinicians often recur to invasive techniques such as open lung biopsy.

In recent years, immunoglobulin levels, immunoglobulin G subclasses, and specific antibody levels have been studied in bronchoalveolar lavage fluid of PBD patients and asymp-

tomatic subjects, but the results have been somewhat contra- dictory (4-6). In addition, the invasive nature of the proce- dure, as well as the difficulties derived from expressing the results so as to allow an estimate of the real concentration of the immunoglobulins, limit its application in a clinical screen- ing approach and in follow-up. Therefore, the development of noninvasive tests that can play a role in the evaluation of patients are needed.

The concept of a common mucosal immune system has been recently enforced (7). It is now clear that a mucosal an- tibody response against local antigenic exposure is shared with other mucosal regions, near or distant. In fact, a lung-con- ducted humoral immune response with systemic and local antibody component can be confidently reflected on related fluids such as saliva. In order to look for alternatives other

Received September 22, 1995; accepted October 23, 1995.

Address reprint requem to Felipe Mendoza Pirez, Instituto Nacional de Enfermedades Respiratorias, ’I lalpan 4502; CP 14080, MBxico D.F., MBxico.

0 1996 Wlley-Liss, Inc.

150 Mendoza et al.

than blood sampling to detect pigeon antigen exposure, we evaluate by enzymatic immunoassay (ELISA) the levels of IgG and IgA antibodies against pooled pigeon serum antigen in both serum and saliva samples. Four groups were included: (1) patients with PBD, (2) asymptomatic individuals exposed to pigeon antigens, (3) healthy subjects who denied pigeon antigens exposure, and (4) patients with idopathic pulmonary fibrosis (IPF), another frequent interstitial lung disease that may be confused with PBD in areas where the prevalence of keeping pigeons at home is high (8). Comparison of levels of antibodies among the four groups were performed.

MATERIALS AND METHODS

Patients

We studied 37 consecutive adult patients attending our in- stitute who were diagnosed of interstitial lung disease. In gen- eral, patients displayed progressive dyspnea, bilateral reticulonodular images on chest roentgenograms, a restric- tive functional pattern, and hypoxemia at rest that worsened with exercise. Exposure to pigeons and other risk factors for pulmonary disease were recorded by a standardized question- naire in the first consultation.

An open lung biopsy was performed for all patients, and subsequently the diagnosis of PBD or IPF was established. The histological findings characteristic of both diseases have been extensively described (2, 8, 10, 11). Briefly, tissue samples of PBD patients showed a diffuse interstitial inflam- mation of mononuclear predominance, mainly lymphocytes, and frequently multinucleated giant cells in terminal and res- piratory bronchioles as well as in the intralveolar walls. Foamy macrophages were seen in the alveolar spaces, and small and loosely arranged granulomas were observed in interstitiurn. There were no changes suggestive of another interstitial lung disease. Morphological findings in IPF included patchy al- veolar septa1 fibrosis and interstitial inflammation consisting mostly of mononuclear cells but also of neutrophils and eosi- nophils. A variable macrophage accumulation was observed in the air spaces, as was hypertrophied reparative alveolar type I1 epithelial cells. Biopsies lacked granulomas, vasculi- tis, microorganisms, and inorganic material by polarized light microscopy.

Asymptomatic Relatives (AR)

Once PBD diagnosis was established, patients were en- couraged to invite first-degree relatives who share epide- miological habitational conditions, including pigeon exposure, to participate by allowing us to take blood and saliva samples.

Clinical Healthy Voluntary Subjects (CHVS)

in which negative status for pigeon contact was confirmed. Subjects were recruited from the hospital lab workers staff

Sera and Saliva Samples Collection

Serum was obtained by conventional venopuncture, cen- trifuged, and stored at -20°C until used. Along with serum samples, unstimulated whole saliva was obtained by direct mouth-tube deposition with an upper volume limit of 10 ml. Saliva was immediately centrifuged and the recovered super- natant was frozen at -20°C until processed.

Pooled Pigeon Sera Antigen (PSA)

A pool of 10 pigeon sera obtained by cardiac punction was used as source of antigens. Protein concentration of this pool determined by Lowry’s method (9) was 30 mg/ml. PSA was aliquoted and stored at -70°C until used.

Detection of Specific Antibodies by Enzymatic Imm unoassay (ELISA)

Both IgG and IgA APSA were determined in serum and saliva samples. The assays were performed as previously de- scribed (5). Briefly, each well in an immunolon microtiter plate (NUNC) was coated with 100 ng of PSA diluted in car- bonate buffer and washed 3x with PBS-Tween 20. Plates were blocked with PBS-Tween containing 0.5% bovine serum al- bumin for l hour and washed 3x with PBS-Tween 20. Patient sera were diluted 1 : 1,500 for the IgG assay and 1:250 for the IgA assay in PBS-Tween containing 0.5% bovine serum al- bumin. Supernatants of saliva samples were analyzed undi- luted as recovered for both the IgG and IgA assays. Under these conditions, each sample was studied in triplicate and plates incubated for 1 hour. Purified goat anti-human IgG or IgA (Fc specific) conjugated with horseradish peroxidase (Cappel) were added and incubated at room temperature for 1 hour. Finally, orthophenylenediamine was added and incu- bated at room temperature for 10 minutes, and the reaction was stopped with H2S04. We included in all plates a positive pool elaborated with four known positive sera or four positive saliva samples as indicated to obtain the variation coefficient.

Specificity of ELISA for Detection of APSA

In order to evaluate possible cross-reactivity between PSA and human sera, three sera known to be positive for APSA and one serum from one CHVS were tested for APSA as fol- lows: (1) untreated serum, (2) after incubation with PSA, and (3) after incubation with human sera from one hipogammaglobulinemic patient (with nonmeasurable IgG by nephelometric assay). These experiments showed a de- crease inAPSA titers > 90% after incubation with PSAin the three sera positive for APSA in respect to their untreated sta- tus. In contrast, when incubated with human serum without immunoglobulins, no change in APSA titers was detected in the three sera positive for APSA when compared with the same untreated sample. No change inAF’SA titers was detected in the CHVS’ serum in any untreated or mhibited status.

Salivary Antipooled Pigeon Sera Antibodies 151

Statistical Analysis A

The descriptive section includes the arithmetical mean, stan- dard deviation, and proportions. Inferential statistics included parametric and nonparametric (Kruskall-Wallis) analysis of variance; when significant, comparisons between individual groups were performed. Correlation analysis was done by both Pearson's and Spearman's tests. We used the 1989 EPISTAT statistical computer program. The significant P level for sig- nificance on initial ANOVA was 0.05; when multiple com- parisons among groups was made, we adjusted the p value with Bonferroni procedure.

RESULTS

Thirty-seven consecutive patients with an initial diagnosis of interstitial lung disease were prospectively studied, but six of them dropped out because they were unable to continue attending the study. The final group included 31 patients: 17 had PBD (14 women and 3 men; mean age 43.2 & 13.01, and 14 had IPF (10 women and 4 men; mean age 48.5 f 10.3). The AR group was foimed by 19 nonsmoking subjects (13 women and 6 nien; nien age 32 f 7 . 3 , whereas the CHVS included 27 nonsmoking normal volunteers (13 women and 14 men; mean age 28.8 k 7.0). All PBD patients were non- smokers, whereas smoking either at the time of diagnosis or previously was registered for 5 out of 14 patients with IPF. In addition, 4 out of 14 IPF patients also reported domestic ex- posure to pigeons.

Figure 1 shows individual optical density (0.D) values; Table 1 shows mean and standard deviation values obtained from serum and salivary IgG in each group, The CHVS group displayed serum 0.D values ranging from 0.002 to 0.15 (mean f SD; 0.024 k 0.033) and saliva 0.D values from 0.01 to 0.041 (0,010 & 0.011); the AR group showed serum 0 .D val- ues ranging from 0.005 to 1.408 (0.255 k 0.471) and saliva O.D. values from 0.003 to 0.486 (0.104 f 0.15 1). In TPF pa- tients, serum 0.D values varied from 0.014 to 1.338 (0.204 & 0.346), and saliva 0.D values from 0.002 to 1.476 (0.22 & 0.447). Both serum and saliva obtained from PBD patients exhibited higher specific 1gG antibody activity. Serum 0.D values range from 0.018 to 2.379 (1.187 k 0.738), whereas saliva O.D. varied from 0.02 to 2.11 (0.801 f 0.447). Com- parison among groups (Table 2) showed that IgG concentra- tion was significantly higher in PBD patients compared to the other groups, ( P < 0.05). No significant differences were found among AR vs. CHVS or IPF groups. A nonparametric Spearman correlation analysis between serum and salivary paired IgG antibody values showed correlation coefficients of 0.54 for CHVS group (P < 0.05) and 033 for PBD groups (P < 0.05). Serum and salivary IgA antibody values are shown in Figure 2. Serum levels in the CHVS group ranged from 0.001 to 0.074 (0.021 +_ 0.021). Although patients with PBD and IPF displayed a significant increase of serum specific IgA antibody when compared to the AR and CHVS groups,

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Fig. 1. Distribution of individual titers of IgG antibodies against pooled pigeon sera obtained by enzymatic immunoassay in clinical healthy volun- tary subjects (CHVS), asymptomatic relatives (AR), pigeon breeder's dis- ease patients (PBD), and idiopathic pulmonary fibrosis (IPF). A, Sera samples; B, saliva samples.

this test did not discriminate between both diseases. PBD patients displayed values ranging from 0.017 to 0.531 (0.169 & 0.183) and IPF patients from 0.02 to 1.1 (0.275 k 0.348, Fig. 2, Table 2). In contrast, IgA antibody determination in saliva samples showed lower values for IPF and AR patients compared with the CHVS group. The PBD group exhibited higher values than the other three groups, reaching statistical significance when compared withAR and IPF (0.21 8 f 0.202 vs 0,101 f 0.072 and 0.065 k 0.066, respectively) (Table 2).

Intergroup comparisons revealed that serum and salivary IgG APSA showed a more discriminant distribution than se- rum and salivary IgA APSA, with a significantly higher mean value for PBD patients than for the other groups (Table 1).

152 Mendoza et al.

TABLE 1. Antibodies Against Pooled Pigeon Sera (APSA)

Serum samples Saliva samples

IgG APSA IgAAPSA IgG APSA I g h APSA Group (n)a Mean SD Mean SD Mean SD Mean SD

CHVS ('27) ,024" ,033 ,021 .021 ,010 ,011 ,161 ,085 AR (19) ,255 ,471 ,025 ,053 ,104 ,151 ,101 ,072

IPF (14) .204 ,346 ,275 ,348 .220 ,447 ,065 ,066

aCHVS = clinical health voluntary subjects; AR = asymptomatic relatives; PBD = pigeon breeder's disease;' IPF = idiopathic pulmonary fibrosis. SD = standard deviation. *Optical density values.

PBD (7) 1.187 ,738 .169 .183 ,801 ,643 ,218 ,202

DISCUSSION

A number of studies regarding the serum and BAL levels of antibody to pigeon antigens have been published. How- ever, although specific antibodies are more frequently found in PBD patients, a certain percentage of exposed but asymp- tomatic subjects may have similar titres (4-6). Several meth- odological variants such as gel precipitation, complement fixation, ELISA, and Western blot have been employed, but none of these antibody detection techniques can definitely differentiate between exposure and disease. The main prob- lem derived from these findings is related to the value of these immunological tests for obtaining a precise diagnosis in pa- tients with interstitial lung disease, mainly in areas in which keeping birds is a common hobby. In other words, an intersti- tial lung disease patient may exhibit coincidental and non- pathogenic exposure to pigeons and specific circulating or BAL antibodies, and the disease can be erroneously diagnosed as PBD. In addition, BAL is a relatively uncomfortable pro- cedure, which in addition has been rigorously standardizcd in only a few places.

In recent years, the concept of a mucosal immune system as rin integrated system has emerged (7). This concept pro- poses that a localized mucosal immune response against an- tigen exposure is shared with other mucosal regions, near or distant. Accordingly, antigen inhalation could induce a lung humoral response that can be confidently reflected on re- lated fluid as saliva.

In this context, an increasing number of reports dealing

with salivary diagnostic tests have been published (12-14). Saliva is a more accessible fluid and easy to collect, which makes it a very attractive noninvasive procedure. Saliva is constituted by a mixture of salivary gland secretions and gin- gival crevicular fluid. The salivary glands produce the greater part of the IgA content, whereas IgG derives largely from the gingival fluid (15).

Our results show that the value distribution obtained in PBD patients for IgG salivary AYSA, closely resembles the distri- bution of serum IgG antibodies and differs significantly from all other groups. Moreover, specific salivary IgG appears to be more discriminative between PBD patients and asymp- tomatic contacts than specific serum IgG. Therefore, this test could play a role in the study of interstitial lung disease pa- tients in whom hypersensitivity pneumonitis provoked by pigeon antigens is suspected. In addition, the search for IgG salivary APSA has some advantages over blood sampling for the screening of antibody prevalence in communities, because this fluid is easy to obtain (16).

Although specific subclass or secretory component identi- fications wcrc not performed, a Spearman correlation analy- sis for paired determinations of serum and salivary IgG antibodies against PSA showed an r value of 0.54. However, whether the IgG salivary APSA are produced by secretory immune system cells or represent serum antibodies diffusion remains a controversial matter.

However, the determination of IgA APSA showed signifi- cantly higher levels in the PBD group than in the CHVS and

TABLE 2. Intergroups Comparison of Titers of Antipooled Pigeon Sera Antibodies (APSA)

Serum samples Saliva samples

Groups compared" IgG APSA IgA APSA IgG APSA IgA APSA

PBD vs. CHVS P<0.05 PcO.05 P<0.05 NS PBD vs. AR P<O.OS P<0.05 Pc0.05 P<0.05 PBD vs. IPF P<0.05 NS PC0.OS P<0.05 AR vs. CHVS NS NS NS NS AR vs. IPF NS P 4 . 0 5 NS NS IPF vs. CHVS NS k 0 . 0 5 NS NS

"PBD=pigeon breeder's disease; CHVS=clinical healthy voluntary subjects; ARasymptomatic relatives; IPF=idi opathic pulmnnay fibrosis. NS=not significant.

Salivary Antipooled Pigeon Sera Antibodies 153

measurement useless. However, in a clinical setting this is not a common handicap, because the differential diagnosis between PBD and CHVS individuals do not represent a real problem.

The finding of elevated levels of specific IgA in controls may be partially explained by the relatively high background values in the IgA assay, which could be attributed to nonspe- cific IgA binding activity in whole undiluted saliva due to the IgA content (> 10 mg/lOO ml) (1 7). Currently, we are analyz- ing the behavior of this assay with diluted saliva including the secretory IgA APSA. In addition, some cross-reactivity resulting from an intestinal immune response to related avian antigens might account for this finding.

In summary, our results support the notion that measure- ment of antipigeon IgG activity in saliva appears to be a sen- sitive immunological test, at least as good as the serological test, to differentiate PBD patients from other ILD and from asymptomatic but exposed subjects. Nevertheless, further re- search including a higher number of patients is highly desir- able in order to evaluate salivary APSA detection as a diagnostic tool in patients in whom PBD is suspected.

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m w 3. Selman M, Chapela R, Salas J, Sansores R, Camllo G, P&ez-Padilla R,

m x

f n=14

116 w

PBD IPF

Fig. 2. Distribution of individual titers of IgA antibodies against pooled pigeon sera obtained by enzymatic imniunoassay in clinical healthy volun- tary subjects (CHVS), asymptomatic relatives (AR), pigeon breeder's dis- ease patients (PBD), and idiopathic pulmonary fibrosis (IPF). A, Sera samples; B, saliva samples.

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