Immunoglobulin Allotypes and Igg Subclass Antibody Response to Pseudomonas Aeruginosa Antigens in Chronically Infected Cystic Fibrosis Patients

Clin. exp. Immunol. (1992) 90, 209-214

Immunoglobulin allotypes and IgG subclass antibody response to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients

T. PRESSLER*, J. P. PANDEYT, F. ESPERSENt, S. S. PEDERSENt, A. FOMSGAARDt, C. KOCH* & N. H0IBYt *Danish CF Centre, Department of Pediatrics, tDepartment of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Denmark and tDepartment of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA

(Acceptedfor publication 6 August 1992)

SUMMARY Chronic Pseudomonas aeruginosa lung infection is the leading cause of death in patients with cystic fibrosis (CF). Poor prognosis correlates with a high number of anti-pseudomonas precipitins and with high levels ofIgG2 and IgG3 anti-pseudomonas antibodies. Reports ofseveral highly significant associations between certain Gm (genetic markers of IgG on human chromosome 14) and Km (k- type light chain determinants on chromosome 2) phenotypes and immune responsiveness to various antigens suggest that allotype-linked immune response genes do exist in man. Furthermore correlation between Gm types and IgG subclass levels has been reported. A group of 143 CF patients were investigated (31 non-infected and 112 chronic infected). The IgG subclass antibodies to three different P. aeruginosa antigens (P. aeruginosa standard antigen (St-Ag), alginate and LPS) were determinated. Immunoglobulin allotypes were determined by haemagglutination inhibition. Sam- ples were typed for Glm(1,2,3, and 17), G2m(23), G3m(5,21), and Km(1,3). Statistical analysis of our data demonstrate that IgG3 anti-pseudomonas antibody levels and Gm markers are related. IgG3 antibody levels to all investigated P. aeruginosa antigens are significantly higher in sera homozygous for Gm(3;5), somewhat lower in heterozygous sera, and significantly lower in sera homozygous for Gm(1,2,17;21). We suggest that genetic differences between the patients may explain the present differences in subclass patterns.

Keywords Gm types IgG subclasses cystic fibrosis P. aeruginosa antigens

INTRODUCTION aggressive infection, accounting for the correlation between Chronic Pseudomonas aeruginosa lung infection is the leading high IgG2 and IgG3 antibodies and poor pulmonary function. cause of death in patients with cystic fibrosis (CF). In the first On the other hand the IgG subclass antibodies have different decade oflife many patients with CF acquire a chronic broncho- biological properties and the differences in the clinical course of pulmonary infection with P. aeruginosa and despite intensive disease may be a result of the particular biological properties of antimicrobial treatment this infection cannot be eradicated. these subclasses. Specific antibodies against P. aeruginosa antigens increase when Considerable evidence from studies ofinbred strains ofmice the infection becomes chronic, and a poor prognosis has been indicates that the immune responsiveness to certain antigens is demonstrated to correlate with a high number of anti-pseudo- controlled by allotype-linked immune response (Ir) genes [8,9]. monas precipitins [1]. Studies on total IgG subclass distribution Reports of several highly significant associations between and specific IgG subclass pattern of antibodies to different P. certain Gm (genetic markers of IgG on human chromosome 14) aeruginosa antigens have revealed a correlation of high IgG2 and Km (k-type light chain determinants on chromosome 2) and IgG3 antibodies with poor lung function and poor clinical phenotypes and immune responsiveness to various antigens condition [2-7]. The interpretation of these findings may be suggest that such allotype-linked Ir genes do exist in man [10]. dual. The antibody pattern may be the result of a particular Furthermore, correlation between Gm types and IgG subclass levels has been reported [11-13]. The four IgG subgroups Correspondence: Tacjana Pressler, Department of Clinical Micro- are encoded by different cistrons. Although each Gm marker is biology, Rigshospitalet, afsnit 8223, Juliane Maries Vej 28,2, 2100 restricted to only one heavy chain subgroup, it is found in some Copenhagen 0, Denmark. and not in all chains of the subgroup. The IgG heavy chain 209 210 T. Pressler et al. cistrons are very closely linked so that various Gm determinants The patient population was divided into four groups, stay together as gene complexes and studies on the inheritance according to the most probable genotypes; homozygotes for ofGm factors have shown that each ofseveral gene complexes is both Glm and G3m markers-one positive for Gm(3) and very stable. In Caucasians IgG1 chains have either Gm(l) and Gm(5), and another positive for Glm(l,17) or Glm(1,2,17) and Gm(17), or Gm(3), but not both markers; and IgG2 chains are G3m(21); heterozygotes for both Glm and G3m markers- either Gm(23) or lack any known Gm factor; IgG3 chains have positive for both Glm(1,2,3,17)/Gm(1,3,17) and G3m(5,21); either Gm(5) or Gm(21) but not both markers [14]. Thus, an and a group heterozygous for GIm markers-Glm(l,2,3,17) or individual can be described as being homozygous or hetero- Glm(1,3,17) and homozygous for G3m-either G3m(5) or zygous for these factors. G3m(21). The purpose of this study was to determine if there is a relationship between subclass-specific antibody levels to different P. aeruginosa antigens and Gm and Km markers in a Antigens population of chronically infected patients with CF. P. aeruginosa standard antigen (St-Ag). Water-soluble antigens were produced from each of 17 serotypes of the MATERIALS AND METHODS International Antigenic Typing Scheme of P. aeruginosa [1]; equal volumes of each were mixed and designated standard CFpatients antigen. Diagnosis ofCF was established on the basis ofabnormal sweat P. aeruginosa alginate. Alginate is a linear heteropolysac- electrolytes and characteristic clinical features. After diagnosis, charide of the two uronic acids and is characteristic for the all CF patients were seen monthly at the Danish CF centre in mucoid capsule of CF strains. It was isolated and purified from Copenhagen. At each visit the clinical condition and sputum mucoid P. aeruginosa [17]. The alginate did not cross-react with bacteriology were examined and have thus been recorded antibodies against lipopolysaccharide or protein antigens of P. prospectively since 1970. The infection was regarded as chronic aeruginosa. A mixture of three serologically cross-reacting if P. aeruginosa had been cultured from sputum at monthly alginates was used [17]. intervals for 6 months. P. aeruginosa lipopolysaccharide (LPS). P. aeruginosa A group of 143 patients from our centre were investigated. strain 1118 0: 3 was isolated from a patient with CF. LPS was This group was a random sample of our entire patient extracted by the phenol-chloroform-petroleum ether method population and includes 31 non-infected and 112 chronically [18] and purified and characterized as reported elsewhere [19]. infected patients. The duration ofchronic infection ranged from This is the most common O-group of P. aeruginosa and it cross- I to 24 years (median 10-8). The age of the patients was evenly reacts serologically with polyagglutinable strains which are distributed in the range 4-40 years (median 18 9 years). All common in CF [20]. serum samples for antibody determinations and Gm and Km allotyping were collected during a period of 1 month. In order to avoid differences in management of chronic P. aeruginosa lung Enzyme-linked immunosorbent assays infection in different time periods we chose a group of CF IgG subclass antibody response was measured by 12 ELISAs patients (n = 77) with duration of a chronic P. aeruginosa that detected IgG subclass antibodies specific for the three infection between 6 and 15 years (median I1 years). This group different P. aeruginosa antigen preparations. Details of the of patients has been admitted every 3 months for a 2-week assays have been published [4,3,21]. course of intravenous anti-pseudomonal treatment since chro- Reagent volumes, blocking and dilution buffer, washing nic infection was established. This treatment regimen has been buffer and washing steps, and development of colour reaction used in our centre since 1976 [15]. were common in all assays. The procedures used are shown in The subclass antibody levels in this group of patients were Table 1. used to evaluate the correlation between Gm markers and anti- Reagent volumes were 01 ml, the dilution buffer was pseudomonas IgG subclass levels. phosphate-buffered saline (PBS, pH 7-2) with 0 1% Tween 20 (Polysorbate, Sigma, St Louis, MO) and 0-5 M NaCl. Between Immunoglobulin allotype each step, plates were washed three times in PBS-Tween. After Immunoglobulin allotypes were determined by haemagglutina- coating with either alginate or St-Ag residual binding sites tion inhibition [16]. Samples were typed for Glm(1,2,3, and 17), were blocked with dilution buffer. Dilutions of serum were G2m(23), G3m(5,21), and Km(1,3). incubated for 1 h in the anti-alginate and anti-LPS assays and

Table 1. Experimental details of ELISAs used to determine antibodies to P. aeruginosa antigens in patients with cystic fibrosis

MoAb dilution Serum dilution Coating anti-IgG conc. Time IgG horseradish peroxidase-conjugated Temp. 0C Antigen Solid phase (mg/ml) (h) 1 2 3 4 1 2 3 4 all steps Alginate MicroWell 30 1 1:500 1:500 1:500 1:250 1:6000 1:2000 1:2000 1:500 35 St-Ag Maxisorb 22 1 1:3000 1:3000 1:3000 1:3000 1:20000 1:3000 1:1000 1:8000 22 LPS Polysorb 10 18 1:2000 1:250 1:2000 1:250 1:5000 1:1000 1:1000 1:500 22shaking Gm types and IgG subclass antibodies in CF 211

overnight in the anti-St-Ag assays. Horseradish peroxidase- RESULTS labelled monoclonal anti-IgG subclass specific antibodies Gm phenotypes (Janssen Biochimica, Belgium, Catalogue number: 24.145.89, The frequencies of the Gm phenotypes observed in the popula- 24.144.88, 24.143.87 and 24.142.86) were added to each well. tion of 143 cystic fibrosis patients are shown in Table 2. Antigen specificity and subclass in antibody specificity these The allotype distribution of Gm phenotypes in our patient assays have been established previously [3,4,22]. Dilutions used population is not significantly different when compared to other are shown in Table 1. The different dilutions were chosen in healthy populations [23,24]. order to obtain the same magnitude of optical density (OD492 = 1 of a standard serum for all in 0) four subclasses all Distribution ofIgG subclass antibodies to P. aeruginosa antigens assays. Sodium citrate 1 M, (0- pH 5-0), containing 1,2-pheny- The median and range of IgG subclass antibodies to three lendiamine-dihydrochloride (2-2 mM) (Sigma) and H202 (6-5 different P. aeruginosa antigen preparations are shown in Table mM) was to added each well, and after incubation in the dark 3. The patients are divided according to the duration ofchronic for 60 min in the anti-alginate ELISAs and 30 min in the anti- infection. Patients without P. aeruginosa infection have low St-Ag and assays. was anti-LPS The enzyme reaction stopped levels of subclass antibodies to all investigated antigens. When by the addition of 1 M was H2SO4. Optical density read at 492 the infection became chronic, IgG subclass antibodies to all nm (OD492) on an automatic plate reader (BioRad, Richmond, investigated antigens increased significantly, but IgG2 anti- CA). bodies to alginate developed more slowly. Only 10-15 years The antigen concentration, serum and antibody dilutions after onset of infection do the IgG2 antibodies to alginate and incubation times were chosen after preliminary investiga- become significantly higher than in non-infected patients. tions with serial dilutions of sera from non-infected and chronic P. aeruginosa-infected CF patients. All samples were tested in duplicate. The assays were calibrated by using a standard of pooled Table 2. Frequency of the Gm phenotypes observed in a of 143 serum from 10 chronically infected patients with CF and with population patients with cystic fibrosis compared to markedly elevated titres of anti-P. aeruginosa antibodies. The healthy controls [24] results are expressed as ELISA units (EU) and were calculated by dividing the mean OD value of the test sample by the mean CF patients Healthy controls OD ofstandard serum having an arbitrary value of 100 EU in all Gm phenotypes number of sera (%) (%) four subclasses. 1,2,3,17;23;5,21 11 (7 7) 9 5 1,2,3.17;. .;5,21 8 (5-6) 8 5 Statistical analysis 1,3,17;23;5,21 23 (16-1) 16-6 Single or two factor analysis ofvarians (Anova) were used when 1,3,17;. .;5,21 15 (10-5) 6 5 more than two groups were compared. The Mann-Whitney U- 1,3,17;. .;21 3 (2-1) test was used for non-parametric unpaired data where two 3;23;5 45 (32 2) 31 2 3;. .;5 19 (12 6) 6-5 groups were compared. Calculations were performed on Apple 1,17;23;21 3 (2.1) Macintosh, using StatViewTM 512 + as software. For the Anova 1,17;. .;21 3 (2.1) tests the data were log transformed before calculations. 1,2,17;. .;21 10 (7 0) 6 5 The level of significance was 5% for a two-tailed compari- Others 3 (2 0) son.

Table 3. IgG subclass antibody distribution to P. aeruginosa antigens

St-Ag LPS Alginate Duration of infection IgG IgG IgG (years) 1 2 3 4 1 2 3 4 1 2 3 4

Non-infected median* 11 0 2 0 3 2 1 0 2 0 5 0 1 n=31 range 2-134 0-10 0-28 0-5 0-218 0-36 0-10 0-5 0-209 0-11 0-42 0-60 1-5 41 6 6 4 17 19 2 2 17 2 0 0 n=16 8-110 0-42 0-70 0-24 2-216 4-89 0-245 0-30 1-115 0-70 0-176 0-5 6-10 74 26 20 20 118 55 37 16 45 1-5 8 1 n=28 7-98 3-117 0-182 0-114 0-590 2-283 0-1140 0-423 0-98 0-118 0-106 0-280 11-15 83 51 55 17 126 90 65 24 65 9 33 3 n=56 33-136 4-163 0-297 0-161 1-714 4-363 0-859 0-453 2-199 0-226 0-340 0-133 > 15 79 41 83 10 68 96 178 38 28 18 19 5 n=12 42-109 30-110 14-183 1-77 16-495 53-322 0-413 11-260 0-246 3-101 0-150 0-31

* ELISA units. 212 T. Pressler et al.

Table 4. The distribution of IgG subclass antibody levels to P. aeruginosa antigens and Gm phenotypes in 77 chronic infected cystic fibrosis patients (results expressed as median of groups)

St-Ag LPS Alginate

Gm IgG IgG IgG phenotypes Number 1 2 3 4 1 2 3 4 1 2 3 4

1,2,3,17;23;5,21 7 61 46 18 20 71 58 16 25 72 11 50 23 1,2,3.17;23;5 1 1,2,3.17;. .;5,21 4 815 74 665 105 113 1315 425 195 28 205 25 3-5 1,3,17;23;5,21 14 82 48 47 5 35 5 150 86 35 30 61 5 11-5 3-5 1,3,17;. .;5,21 7 70 36 33 6 142 5 76 5 32 7 65 8 19 3 1,3,17;..;21 3 73 40 5 0 139 120 4 1 22 0 0 10 1,3,17;23;21 1 3;23;5 19 79 52 78 9 96-5 67-5 130-5 16 77 11 60 0 3;. .;5 8 83 55 30 13 74 63 5 16 5 17 5 37 12 21 2 1,17;. .;21 3 77 31 13 47 283 68 65 91 50 3 0 119 1,17;23;21 1 1,17;23;5,21 1 1,2,17;. .;21 8 92 5 27 21 18 119 5 56 19-5 17-5 48 1 5 5.5 0 5

Table 5. Relationship for IgG subclass antibodies to P. aeruginosa antigens and Gm genotypes, Gm(23) markers and Km types (results expressed as median of the groups)

St-Ag LPS Alginate IgG IgG IgG Genotypesa Number 1 2 3 4 1 2 3 4 1 2 3 4

Homozygote Gm(3;5) 27 79 5 53.5 51-5* 11 96 5 63 5 91.5* 16 57 11 39* 2 Heterozygote for Glm and G3m 32 77 41 5 30 19 119 85 30 5 19 5 62-5 8 5 19 5 3 5 Homozygote Gm(1,2,17;21) 12 88 5 29 5 17 5 25 183 63 37-5 41 50 2 3 5 3 Miscellaneous 6 75 5 39 24 13 5 149 5 125 5 33 5 19 54 0 23-5 7 Gm(23) pos 44 79 50 48* 20 110 63 57* 25 69* 7 36* 3 neg 32 80 38 21 13 139 80 21 16 41 5 11 3 Km 1,3 11 73 23 11 16 64 32 45 12 36 0 10 0 Km 3 66 79 48* 40* 19 121 69 38 24 50 9* 24 3

*P< 05. a The groups refer to definitions in Materials and Methods.

Gm allotypes and specific subclass antibody levels and significantly lower in sera homozygous for Gm(1 ,2,17;21). The median immune responses to P. aeruginosa St-Ag, alginate When G2m(23) factor influences on subclass antibody levels and LPS in relation to various Gm phenotypes are shown in were calculated separately, we found that persons positive for Table 4. The patient population could be divided into 13 Gm(23) had higher levels of IgG3 antibodies to all P. aeruginosa different phenotypes. Four phenotypes including only one antigens investigated, and higher IgGl antibodies to alginate patient have been excluded from the statistical calculations. than patients negative for this marker (Table 5). The influence of There were no significant differences in IgG subclass levels Km markers for subclass antibody levels was calculated and we according to Gm phenotypes. There were no significant differ- found that homozygotes for Km(3) had higher level of IgG2 to ences between groups according to age of the patients or to P. aeruginosa St-Ag and alginate. IgG3 to St-Ag were also duration of chronic infection (data not shown). higher in homozygotes compared to heterozygotes for Km(3). Data on the distribution of IgG subclass antibodies to P. In our population we did not find any Km(l) homozygotes, aeruginosa antigens according to the homozygous/heterozygous which is not surprising because of the rarity of the Km(l) allele status for Glm and G3m are shown in Table 5. IgG3 antibody [25]. levels to all investigated P. aeruginosa antigens are higher in sera Testing of interactions between Gm and Km markers homozygous for Gm(3;5), somewhat lower in heterozygous sera showed no interactive effect between these markers. Gm types and IgG subclass antibodies in CF 213

DISCUSSION We found the same correlation when total IgG3 concentration was investigated in this group of patients (P=0-006) (unpub- The chronic P. aeruginosa infection causes a pronounced lished data). specific antibody response in patients with CF and a poor Our investigations provide evidence for a genetic control of prognosis has been associated with an increasing number of the capacity of a given individual to respond to an antigen by anti-pseudomonas precipitins [1]. The course of the infection producing antibodies of preferably one or another IgG subclass. differs, however, individually. In a previous study we found an Our results support the hypothesis that regulatory elements association between elevated serum levels of P. aeruginosa linked to the Gm(3;5) haplotype result in preferential switching specific IgG3 immunoglobulin and poor lung function. Patients to the IgG3 rather than to other Ig heavy chains as suggested by with a rapid downhill course of the clinical condition and Hassan et al [13]. progressively increasing pulmonary destruction showed rapid In this study we investigated a group of patients with the increase of IgG2 and IgG3 P. aeruginosa specific antibodies. same genetic disorder [37], resulting, probably from the defec- Evidence has been accumulating to suggest a contributing role tive chloride channel protein CFTR which leads to increased ion of antibodies to P. aeruginosa antigens in the progressive lung concentration in the sweat and in the respiratory fluid [38], in an damage in CF in which tissue damage is due to immune-complex unusual predilection of the patients to chronic P. aeruginosa mediated inflammation leading to lung injury [26,27]. respiratory tract infection. From a clinical point of view the IgG subclasses differ from one another both immunochemi- chronic P. aeruginosa infection runs a high variable course in cally and functionally and current knowledge is incomplete with individual patients [39]. Some patients remain in good condition regard to the roles of the four subclasses of IgG in human health with only slight deterioration in pulmonary function over a and disease. Complement activation through binding of Clq is period of many years, while other patients display a rapid most effective with IgG1 and especially IgG3 proteins. The downhill course with progressive pulmonary destruction. We binding to macrophage receptors is most active with IgG1 and suggest that genetic differences between the patients may IgG3 proteins [28-30]. explain the present differences in subclass patterns, and that the In the present study we report a significant association unfavourable clinical course seen in patients with high anti- between Gm(3;5) phenotype and high IgG3 antibody levels to P. pseudomonas IgG3 level may be a consequence ofthe particular aeruginosa antigens. The data reported here do not elucidate biological properties of these subclasses [28-30]. whether the immune response is controlled by Gm loci or by loci closely linked to them. The immune response is based on ACKNOWLEDGMENTS cellular and molecular each of which is complex interactions, We thank Lisbeth Heiden for excellent technical assistance. This study subject to the control mechanisms related to allelic polymor- was supported by grants from Ville Heise Foundation and Danish phism of the molecule and gene concerned. The genes that show Hospital Foundation for Medical Research, Region of Copenhagen, allelic polymorphism and regulate immune responses to particu- The Faroe Islands and Greenland. lar antigens have been called immune response (Ir) genes. Some of these Ir genes are closely linked to other polymorphic genes REFERENCES whose products bear allotypes. One category of well-known Ir 1 H0iby N. Pseudomonas aeruginosa infection in are ones. Furthermore, in humans, cystic fibrosis. genes Ig allotype-linked Diagnostic and prognostic significance of Pseudomonas aeruginosa antibody responses to some microbial antigens have been precipitins determination by means ofcrossed immunoelectrophore- reported to be under the control of Ig allotype (Gm, Km or sis. 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