Vol. 30, No. 8

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2013-2018

0095-1137/92/082013-06$02.00/0 Copyright X 1992, American Society for Microbiology

Immunoglobulin A (IgA) and IgG Serum Antibodies to Mycobacterial Antigens in Crohn's Disease Patients and Their Relatives LAWRENCE G. WAYNE,'* DANIEL HOLLANDER,2 BEATRIZ ANDERSON,1 HILDA A. SRAMEK,1 CONSTANCE M. VADHEIM,3 AND JEROME I. ROTrER3 Tuberculosis Research Laboratory, Department of Veterans Affairs Medical Center, Long Beach, California 908221; Gastroenterology Division, Department of Medicine, California College of Medicine, University of California, Irvine, California 927172; and Division of Medical Genetics, Departments of Medicine and Pediatrics, Cedars Sinai Medical Center, University of California, Los Angeles, California 900483 Received 4 February 1992/Accepted 18 May 1992

Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies to Mycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responses to mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patients and their relatives had significantly higher IgG responses against these antigens than did the controls. The elevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it was against soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under the loosely bound peptidoglycolipid or glycolipid antigens of mycobacteria. An etiologic role for mycobacteria in Crohn's disease has long been the subject of study and speculation. Interest in this possibility was stimulated by recent reports of the isolation of mycobactin-dependent mycobacteria from the intestinal tissues of patients with Crohn's disease (2, 3). These organisms have been shown by nucleic acid analyses to be indistinguishable from Mycobacterium paratuberculosis (15, 33), the agent of Johne's disease, a granulomatous enteritis of ruminants. However, these mycobactin-dependent strains have been isolated from only a small proportion of human bowel specimens examined, and evidence for the presence of a variety of other mycobacterial species has been found in the tissues of patients with Crohn's disease (6, 8, 34). This calls into question the specificity of the possible etiologic role of these bacteria and suggests that such a role, if it does exist, may belong to environmental mycobacteria, in general, rather than to a single species. With this in mind, we undertook a serologic survey of patients with Crohn's disease, as well as their blood relatives, their spouses, and unrelated control subjects. The sera were examined for antibodies of both the immunoglobulin A (IgA) and IgG types to whole cells of three species of mycobacteria from which the loose outer envelope of serovar-specific antigen (1) was removed and to crude protein extracts of sonically disrupted mycobacteria. The mycobacteria selected for study were the pathogen M. tuberculosis, which is not found free in the environment; M. avium, which is an environmental opportunistic pathogen; and M. gordonae, which is a common environmental organism that is rarely, if ever, the cause of disease (25, 28).

TMC 1324, all of which are maintained in the Long Beach laboratory collection. Limited additional studies were also done with M. paratuberculosis Linda, which was provided by Roderic Chiodini, and strains of M. avium and M. intracellulare representing various serovars, which were provided by Anna Tsang. The mycobacteria were grown in Dubos broth base (Difco Laboratories, Detroit, Mich.) containing 1% (wt/vol) glycerol and were enriched with Dubos medium albumin (Difco) for M. tuberculosis and M. gordonae or with Dubos oleic albumin complex (Difco) for M. avium and M. intracellulare. For growth of M. paratuberculosis, the medium was also supplemented with 2 ,ug of mycobactin J (Allied Laboratories, Ames, Iowa) per ml. The methods used to grow and harvest the mycobacteria have been described previously (27). Two types of antigens were used, isopentanol-extracted formalin-fixed whole cells and crude, protein-enriched sonic extracts of the bacilli. Salcedo et al. (21) reported that isopentanol can extract polar lipids such as nonionic detergents from protein solutions without denaturing or otherwise damaging the proteins. We used this solvent to remove the loosely bound serovar-specific polar peptidoglycolipid cell wall sheaths (1) and to expose the fixed cell surfaces of the mycobacteria for use as whole-cell antigens. Samples of 200 ,ul of 10% (vol/vol) aqueous suspensions of formalin-fixed mycobacterial cells were mixed vigorously with 10 ml of isopentanol and centrifuged (900 x g, 30 min). The supematant solvent was discarded, and the isopentanol-treated cells were washed once in 5 ml of aqueous 0.02% Tween 80 and resuspended in water to a concentration of approximately 10 mg of moist cells per ml. The method for preparation of crude protein-enriched sonicates has been described previously (27). One study was carried out with baker's yeast (Saccharomyces cerevesiae ATCC 7754), which was provided by Ruth Russell. The yeasts were grown and the whole cells were fixed for use as an antigen as described by Main et al. (14). Human sera. Sera from the following categories of human

MATERIALS AND METHODS Antigens used. Most of the studies were done with wholecell antigens prepared from M. tuberculosis H37Rv, M. avium SJB-2, M. intracellulare TMC 1403, and M. gordonae *

Corresponding author. 2013

2014

WAYNE ET AL.

subjects were examined (the average + standard deviation age in years is given for each group): patients with Crohn's disease (43.5 + 16.5), blood relatives of patients with Crohn's disease (45.9 + 18.0), spouses of patients with Crohn's disease (48.0 + 13.8), and normal Red Cross blood donors and other unrelated control subjects (42.6 + 16.1). The criteria for inclusion of patients with Crohn's disease and their relatives were the same as those described for prior studies (10, 12). Sera and clinical information were collected under the guidelines of the human studies committees of our respective institutions, and the privacy of the information was protected. Rabbit sera. Antisera raised against unextracted cells of M. avium complex agglutination serovars 1 to 9, 12, 14, 15, 18 to 20, 23, and 25 were provided by Anna Tsang. The sera against unextracted cells of M. paratuberculosis Linda were raised in the Long Beach laboratory by methods described by Good and Beam (7). ELISAs. For enzyme-linked immunosorbent assays (ELISAs) with isopentanol-treated whole-cell antigens, the suspensions were treated briefly in a low-energy Bransonic 3 cleaning bath (Heat Systems-Ultrasonics, Farmingdale, N.Y.) to disrupt clumps without breaking the cells. Fortymicroliter volumes of the cell suspensions were dispensed to wells of flat-bottom NUNC II ELISA plates (Vangard International, Neptune, N.J.) and were incubated overnight (uncovered) at 37°C to dry. On the following day, 50 ,ul of phosphate-buffered saline, (pH 7.5) containing 100 ,ug of thimerosal per ml (PBSM) and 50 Ru of 0.05 M bicarbonate buffer (pH 9.4) were added to all wells and the plates were incubated at room temperature for 30 min. The buffer was then aspirated, and the wells were blocked for 2 h at 37°C with 2% normal equine serum in PBSM. The plates were washed with a solution of 200 ,ug of bovine serum albumin per ml in PBSM and could be used immediately or after dry storage at 5°C for 2 weeks or more. For tests with crude protein antigens, the wells of the plates were primed overnight with 50 ,ul of a solution containing 40 ,ug of the desired protein per ml in PBSM and 50 ,Il of 0.05 M bicarbonate buffer (pH 9.4). For human antibody assays, 100-,ul samples of the test subjects' sera, diluted 1:100 (for IgG) or 1:20 (for IgA) in PBSM, were dispensed to wells coated with the selected antigens and to a blocked well without antigen. After 2 h of incubation at room temperature, the serum was aspirated and the wells were washed three times with 0.05% Tween 20 in normal saline and once in saline without Tween 20. One hundred microliters of second antibody, peroxidase-conjugated immunoaffinity-purified goat anti-human IgG or IgA (Sigma Chemical Co., St. Louis, Mo.) diluted 1:400 in 10% equine serum in PBSM, was dispensed to each of the test wells, and the wells were incubated at room temperature for 2 h. After aspiration of the second antibody, the wells were washed four times with Tween 20-saline and once with PBSM. After the addition of 200 ,ul of a freshly prepared solution of 0.33 mM 2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (Sigma) and 0.34 mM H202 in 0.1 M acetate buffer (pH 4.6) to each well, the plates were incubated in the dark for 2 h at room temperature. The reaction was stopped with 10 ,ul of 1 M oxalic acid, and the optical A405 was measured on a model EL307 ELISA reader (Bio-Tek Instruments, Burlington, Vt.). For assay of rabbit antibodies, the procedure was performed as described above, except that the dilutions of antibody to the different serovars were based on their known agglutinating titers, and the second antibody was peroxi-

J. CLIN. MICROBIOL.

dase-conjugated goat anti-rabbit IgG (ICN Immunobiologicals, Lisle, Ill.). The readings for each specimen were corrected for nonspecific background by subtracting the A405 of the corresponding antigen-free well from those of the antigen-primed wells. When theA405 of a sample was off scale on the reader, i.e., greater than 2.0, half of the sample in the well was transferred to an empty well; the sum of the A405 of the residual fluid in the original well and that of the transferred aliquot represented the ELISA score for that sample. The commercial goat anti-human and anti-rabbit globulins that were used as peroxidase conjugates for second antibodies were produced with Freund's adjuvant, which contains killed mycobacteria. To ensure that false-positive reactions were not produced in our tests by antimycobacterial antibodies in these conjugates, control wells in the ELISA plates were primed with the mycobacterial antigens and challenged with the second antibody without exposure to human or rabbit serum. No reactions with A405 values higher than those of the substrate blanks were seen between the antigens and the second antibodies with the reagent concentrations used for these tests. Statistical tests. Statistical significances of the differences between distributions of raw scores for serum among the human subjects were determined by the nonparametric rank-order Mann-Whitney U test (22). Correlation coefficients were calculated with the Sigmaplot version 3.10 program (Jandel Scientific, Corte Madera, Calif.).

RESULTS In a preliminary experiment (data not shown), suspensions of unextracted cells of a strain each of M. avium complex agglutination serovars 1 to 9, 12, 14, 15, 18 to 20, 23, and 25 and of M. paratuberculosis Linda were dotted onto nitrocellulose membranes and tested against homologous and heterologous rabbit antisera by using peroxidase-conjugated goat anti-rabbit IgG and hydrogen peroxide-diaminobenzidine reagent (24) to detect antibody binding. None of the antisera to the M. avium complex serovars gave positive reactions to the M. paratuberculosis cells, but antisera to M. paratuberculosis appeared to react as strongly to the cells of the other selected strains as they did to their homologous strain. This suggested that the dominant antibody in the anti-M. paratuberculosis serum is to an underlying nonpeptidoglycolipid surface antigen that is common to the entire complex. To test this hypothesis, unextracted and isopentanol-extracted whole cells of M. intracellulare TMC 1403 serovar 14 were assayed by ELISA against homologous and heterologous serovar rabbit antisera by using peroxidase-conjugated antirabbit IgG as the second antibody. Isopentanol extraction caused an average of 72% reduction in the ELISA score of homologous serovar reactions against whole cells, whereas extraction had little or no effect on most of the heterologous reactions (Table 1). When rabbit antisera raised against unextracted cells of M. paratuberculosis Linda were tested against isopentanol-extracted and unextracted cells of the homologous strain, the isopentanol-treated cells gave slightly higher responses than did the extracted cells (ratio of the A405 of isopentanol-treated to A405 of untreated cells = 1.16). These results demonstrated that isopentanol extraction removes the loosely bound serovar-specific peptidoglycolipid surface antigen and exposes a common surface antigen on cells of the M. avium complex, and that even the serovar-specific antisera have some antibody to that underlying antigen. Our antibody that was raised against the

MYCOBACTERIAL ANTIBODY IN CROHN'S DISEASE

VOL. 30, 1992

2015

TABLE 1. ELISA of unextracted and isopentanol-extracted cells of M. intracellulare serovar 14 against selected rabbit antisera to homologous and heterologous serovars Relationship

Homologous

Batch no.

567 756 806 1294

Rabbit serum Serovar or

A405 against serovar 14 cells

strain

Dilution Dlto

nxrce

(A)

14 14 14 14

1:1,280 1:320 1:640 1:1,280

2.750 2.781 1.074 2.406

A

xrce

0.953 0.833 0.269 0.513

Mean ± SD

Heterologous

(B) B

Ratio (B/A)

0.35 0.30 0.25 0.21

0.28 ± 0.06

692 500 1094 1240 159 160

6 7 7 8 Linda Linda

1:640 1:640 1:640 1:1,280 1:400 1:200

Mean ± SD

unextracted whole cells of M. paratuberculosis appears to be directed to that common antigen. M. paratuberculosis has been shown by nucleic acid homology to belong in the species M. avium (15, 33). The demonstration of the common surface antigen reactivity led us to conduct our human serum survey with isopentanol-treated cells of M. avium instead of M. paratuberculosis, which is difficult to grow in quantity. When isopentanol-treated whole mycobacterial cells were used as antigens and the human sera were tested for antibody of the IgG class, the reactions of sera from patients and their relatives and spouses were not significantly different from one another, but they were significantly higher than those of the unrelated control group (Fig. 1A, C, and E) for all mycobacterial antigens used. On the other hand, when the sera were tested for antibody of the IgA class, the reactions of sera from patients with Crohn's disease against the same three whole-cell antigens (Fig. 1B, D, and F) were significantly elevated over those of sera from all other subject groups, including the relatives of patients with Crohn's disease. This experiment was repeated with the same M. avium isopentanol-treated whole-cell antigen and was also done with a comparable antigen prepared from M. intracellulare TMC 1403 (data not shown); again, the distributions of IgA scores of the patients with Crohn's disease were significantly higher (P < 0.01) than those of their relatives. Species-versus-species regression analyses of the IgA ELISA scores with the whole-cell antigens were run for the M. avium, M. tuberculosis, and M. gordonae antigens (Table 2). Among the two most reactive groups of human subjects, i.e., the patients with Crohn's disease and their relatives, the mean IgA ELISA scores were greatest against M. avium and lowest against M. gordonae. Among these two subject populations, the correlation coefficients between paired antigens ranged from 0.61 to 0.90; the highest correlations were seen between the responses to the M. avium and M. gordonae antigens with both the patients with Crohn's disease (r = 0.89) and their relatives (r = 0.90). When soluble protein extracts of either M. avium or M. tuberculosis were used as antigens, nonsignificant (P > 0.05)

1.006 1.841 0.339 1.118 3.707 3.766

0.487 1.753 0.348 1.135 3.562 3.577

0.48 0.95 1.03 1.02 0.96 0.95

0.90 + 0.21

or borderline significant (P > 0.03) differences in the IgG responses were seen among all groups of subjects. The M. avium-soluble antigen yielded IgA scores (data not shown) with sera from patients with Crohn's disease that were low (median A405, 0.104) but significantly higher than those of their relatives (median A405, 0.054; P = 0.007) or the normal controls (median A405, 0.031; P = 0.0003); no significant difference was seen between the relatives and the normal controls. With M. tuberculosis soluble antigen, a very small, but significant (P = 0.012) elevation in the median IgA score was seen in patients with Crohn's disease over the normal controls, but no difference was seen between these patients and their relatives. When whole baker's yeast cells were used as the antigen, the sera from patients with Crohn's disease exhibited significant elevations of both IgG (P = 0.011) and IgA (P = 0.0025) ELISA scores over those seen with serum from their relatives (Fig. 2), as well as those seen with sera from the 10 other control subjects, which comprised the patients' spouses and other healthy individuals (P < 0.025) (data not shown in Fig. 2); the healthy relatives' responses were not significantly different from those of the other controls. When the IgG reactions to yeast cells of serum from patients with Crohn's disease were compared with their IgA reactions against that antigen, a high correlation (r = 0.84) was seen. On the other hand, a similar comparison between IgG and IgA reactions with the M. avium antigen yielded a much lower correlation (r = 0.46).

DISCUSSION The data from the present study indicate that sera both from patients with Crohn's disease and from their relatives exhibit higher IgG ELISA scores to fixed mycobacterial surface antigens than do those of normal control subjects. This could be due to increased environmental exposure to the bacilli or increased serological response to a common environmental exposure. An environmental factor must be considered in view of the significant elevations in antimycobacterial IgG seen in sera from the small number of patients' spouses in this series. However, this factor will have to be

WAYNE ET AL.

2016

J. CLIN. MICROBIOL.

IgG

IgG

IgA :

IgA

.5 -

C

A

D I

1.0-

.

to

0.5 -

*0 .0

9

to

0

0

o

0.5-

0.5-

0

*O

.

0.25

ol%_

0

0.25-

*.e

S-4 i 0

0

0%

V

.000

0-

0-

CR0

(n=22 CRO REL

REL l SPO

NOR

n-24l(n=5)$n=20'

,SPO NOR

CRO REL SPO NOR (n=21) n=24) (n=5) (n=21) CRO REL .032N SPO N NOR .000Y N

N

-

IgAA

IgO

o.1 A E

F

0.5 -

1.0-

00 0

I0 00

10

00*

el

A11

0

11 _

4t

CRO REL SPOI NOR n=22) n=24) (n=5)( n=20)

-

L 00

*00 10

O-I

CR0 REL SPO NOR (ns2l) (n=24) (n=5) (n=20)

FIG. 1. Serologic response of serum from four groups of human subjects to whole-cell antigens of three species of mycobacteria. The top shows the distribution of individual ELISA scores (A40) with median bars, and the bottom shows the significance (P) of the differences in score distributions among the groups as determined by the nonparametric Mann-Whitney rank order test. N, not significant, i.e., P > 0.05. (A and B) M. tuberculosis whole-cell antigen; (C and D) M. avium whole-cell antigen; (E and F) M. gordonae whole-cell antigen. (A, C, and E) Sera tested at a 1:100 dilution for IgG; (B, D, and F) sera tested at a 1:20 dilution for IgA. CRO, patients with Crohn's disease; REL, blood relatives of patients with Crohn's disease; SPO, spouses of patients with Crohn's disease; NOR, unrelated normal control subjects.

0

0

Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives.

Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent as...
1MB Sizes 0 Downloads 0 Views