Feb 3, 1976 - sputum of patients with cystic fibrosis were investigated by using an indirect immunofluorescent technique. Fluorescein-conjugated ...
INFMCTION AND IMMUNITY, July 1976, p. 114-117 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 1 Printed in U.SA.
Interactions of Pseudomonas aeruginosa with Immunoglobulins and Complement in Sputum SUHUNG HANN* AND DOUGLAS S. HOLSCLAW Department of Pediatrics, Hahnemann Medical College and Hospital, Philadelphia, Pennsylvania 19102
Received for publication 3 February 1976
The interactions of Pseudomonas aeruginosa with humoral factors in the sputum of patients with cystic fibrosis were investigated by using an indirect immunofluorescent technique. Fluorescein-conjugated, monovalent antiserum specific to heavy chains of human immunoglobulin A (IgA), IgG, or IgM and to complement C3 were used. All strains of P. aeruginosa recovered from the sputum specimens of patients with cystic fibrosis were found to be coated with antibodies of IgA, IgG, and IgM classes and with C3. The specificity of the antibody coating was determined. The fluorescence was most intense with IgA and was followed in intensity by IgG, IgM, and C3. No difference was noted between rough and mucoid strains of P. aeruginosa. When the subcultured P. aeruginosa was incubated with the sputum eluates, a similar pattern of fluorescence was demonstrated, indicating that these humoral factors are present in the sputum and that the coating process can take place in the lower respiratory tract of the patients. By single radial immunodiffusion, significant quantities of the humoral factors in the sputum eluates were detected. These findings suggest that P. aeruginosa is opsonized in sputum of patients with cystic fibrosis.
Cystic fibrosis (CF) is one of the most common hereditary diseases of children and is characterized by pancreatic deficiency, chronic pulmonary disease, and elevated sweat electrolytes. The basic biological defect is as yet unknown. Among other features, persistent colonization by Pseudomonas aeruginosa of the lower respiratory tract has become the leading cause of pulmonary morbidity and consequent mortality of patients with CF (8, 9). The unique association of P. aeruginosa with CF has not been satisfactorily explained. Systemic invasion by the organism, however, is extremely rare in these patients, which suggests that an immunological defect might exist locally in the lung. Thus far, secretory antibodies in the respiratory tract have been found to be normal in these patients (7, 12, 16). However, a specific serum deficiency in supporting opsonization and phagocytosis of P. aeruginosa has been demonstrated (1, 2). Whether this is due to a defect in immunolgobulin A (IgA) antibodies (1), to the presence of a labile inhibitor (2), or to some other factor is yet to be clarified. Recently, Reynolds showed that IgA, IgM, and IgG and complement components C3 and C4 are present in sputum from patients with CF (14). We have confirmed these findings and, further, have undertaken an indirect immunofluorescent study to investigate the interactions of P. aeruginosa with these humoral factors in sputum.
(This work was presented, in part, at the 16th Cystic Fibrosis Club Meeting, 15 April 1975, Denver, Colo.) MATERIALS AND METHODS Thirty-four fresh sputum specimens were collected in sterile containers immediately after chest physiotherapy from 18 patients with CF and 2 nonCF patients with chronic pulmonary infection. Specimens that contained obvious blood or saliva were considered unsuitable and excluded from the study. Preparation of sputum. Two separate cultures were made from each sputum specimen. Direct smears were made for Wright and Gram stains. A portion of sputum was taken from the center of each specimen, diluted in 3 volumes of phosphatebuffered saline, pH 7.3, vigorously agitated on a Vortex mixer, and sedimented by gravity for 5 min. The bacteria-rich supernatant was taken, washed three times in phosphate-buffered saline, and submitted for immunofluorescent study. Immunofluorescent studies. The indirect immunofluorescence technique described by Thomas et al. (17) was slightly modified as follows. From the washed bacteria-rich supernatant six aliquots of 0.5 ml were centrifuged for 10 min at 1,700 x g, and the supernatant was discarded, leaving 0.1 ml of sputum sediment in each tube. To each tube was added 0.1 ml of 1:4 dilutions of monovalent fluoresceinconjugated rabbit antisera specific to the heavy chains of human IgG, IgA, and IgM and to complement C3 (Behring Diagnostics, Sommerville, N.J.). Because of the possibility that the fluorescein-conjugated antisera may contain antibodies to certain pathogens, antisera were absorbed with subcultured 114
VOL. 14, 1976 P. aeruginosa three times before the experiment. After a 30-min incubation at room temperature and three washings with phosphate-buffered saline, the sediments were examined for fluorescent staining of the bacteria using a fluorescent microscope (AO vertical fluorescence model 2070) at x 1,000 magnification. The intensity of fluorescnce was recorded as negative (-) or positive with gradations of + to
Control for immunofluorescence. Specific blocking of fluorescence was shown by prior incubation of P. aeruginosa with unconjugated, monospecific antisera to human IgG, IgA, and IgM and complement C3. Also, the fluorescein-conjugated antisera were shown to be free of antibodies against subcultured P.
P. AERUGINOSA Ig AND C IN SPUTUM
115
TABLE 2. Fluctuations of intensity of immunofluorescence of P. aeruginosa in sputum specimens collected on different days from CF patients Sources Dates of Intensity of fluorescence against: of collection sputum y-Chain a-Chain ,u-Chain C3
N.M.
R.H.
6-26-74 7-2-74 7-22-74 7-30-74 9-6-74 10-2-74 11-8-74 11-13-74
++++ ++++ ++++ +++ ++++ +++ ++++ ++++
++++ ++++ ++++ +++ ++++ ++++ ++++ ++++
+++ ++ +++ +++ +++ +++ ++ +++
+ +++ + +++ ++ + ++
+ aeruginosa. In vitro coating of subcultured P. aeruginosa. The bacteria subcultured from sputum, which were shown to be free of immunoglobulin and comple- tors in the sputum, sputum eluates were prepared as ment coatings, were first incubated with sputum follows. A portion of sputum was diluted in 3 voleluates for 30 min at room temperature at the rate of umes of normal saline, agitated on a Vortex shaker 106 organisms/0.5 ml of eluate, washed three times vigorously for 5 min, allowed to eluate for 1 h at with phosphate-buffered saline, and then submitted room temperature with occasional shakes, and centrifuged for 20 min at 6,000 x g. The supernatant for immunofluorescent study. Specificity of antibody coating. To demonstrate was then concentrated to its original volume by the specificity of antibody coating in vitro, the spu- filtration through Minicon B-15 with a 15,000tum eluate was first incubated with subcultured P. molecular-weight cut-off (Amicon Corp., Lexington, aeruginosa using about 1010 bacteria/0.5 ml of eluate Mass.). The eluates thus prepared were submitted for 1 h at room temperature. As a control, the same for single radial immunodiffusion plates (Behring procedure was carried out with Staphylococcus au- Diagnostics, Sommerville, N.J.). reus. The absorbed eluates were separated by centrifugation at 6,000 x g and reincubated with 106 of RESULTS the respective bacteria alternately for 30 min at Sputum specimens from all but one patient room temperature. Then the bacterial button was taken, washed three times in phosphate-buffered with CF and one of the two non-CF patients saline, and submitted for immunofluorescent study. with chronic pulmonary infections grew P. Immunodiffusion. To quantitate the humoral fac- aeruginosa, most of them being mucoid strains.
Other pathogens grown in these specimens were S. aureus, group A beta-hemolytic Streptococcus, Klebsiella pneumoniae, and Candida albicans. A Wright stain of sputum smears Patient Intensity of fluorescence against: showed predominantly neutrophilic granulocytes (>95% of cellular elements) and a small Age Sex y-Chain c-Chain A-Chain C3 No. (years) number of mononuclear cells, with over 90% of overall viability by trypan blue exclusion. A 1 17 M ++++ ++++ ++ ++ majority of these granulocytes were found to 2 8 F ++++ +++ +++ +++ contain gram-negative rods in the cytoplasms. 3 24 F +++ ++++ ++ +++ Results of the immunofluorescent study of P. 4 23 F +++ ++++ ++ ++ 5 11 M +++ ++++ ++ aeruginosa in sputum are shown in Tables 1 +++ 6 5 M +++ ++++ ++ +++ and 2. All strains of Pseudomonas recovered 7 20 M +++ +++ +++ + from sputum showed fluorescence with fluores8 14 F ++++ +++ +++ +++ cein-conjugated antisera to human IgG, IgA, 9 24 M ++++ ++++ +++ ++ IgM, and C3. No difference was noted between 10 22 M ++++ ++++ +++ + rough and mucoid strains in this regard. The 11 21 F ++++ ++++ +++ +++ pattern of fluorescence was smooth, linear, and 12 19 M + ++++ + + uniform, surrounding the bacterial surface. In 13 20 M ++++ ++++ +++ +++ general, fluorescence with IgA was most in14 13 M +++ ++++ +++ + 15 17 M +++ +++ tense, followed by IgG, IgM, and C3 (Table 1). ++ ++ 16 12 F +++ +++ +++ +++ Minor fluctuations of fluorescence were noted 17 12 F ++ +++ ++ + among the specimens collected on different 18" +++ 8 M ++++ +++ +++ ydays from the same patient (Table 2). a The bacteria subcultured from sputum, Non-CF patient with chronic pulmonary infecwhich were initially shown to be free of immution. TABLE 1. Immunofluorescence of P. aeruginosa in sputum in CF patients
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HANN AND HOLSCLAW
TABLE 3. Demonstration of specificity of antibody coating Intensity of fluorescence against: Incubation with:
Subcultured bacteria
P. aeruginosa
S. aureus
Phosphate-buffered saline Eluate absorbed with P. aeruginosa Eluate absorbed with S. aureus Unabsorbed sputum eluate Phosphate-buffered saline Eluate absorbed with S. aureus Eluate absorbed with P. aeruginosa Unabsorbed sputum eluate
noglobulin and complement coatings, exhibited fluorescence with these antisera after incubation with sputum eluates. The pattern of fluorescence was very similar to that of the original bacteria. The specificity of antibody coating is shown in Table 3. P. aeruginosa was found to be coated with specific antibodies of IgG, IgA, and IgM classes. The results of immunodiffusion on 11 sputum eluates from CF patients are as follows. IgG was present in the highest quantity, with a mean of 55.92 mg/dl (standard deviation [SD] ± 27.82 mg/dl), followed by C3, with a mean of 22.82 mg/dl (SD ± 7.61 mg/dl). The mean level of IgA was 13.77 mg/dl (SD + 7.91 mg/dl), and IgM was present in the lowest quantity, with a mean of 1.75 mg/dl (SD + 1.98 mg/dl).
DISCUSSION
Opsonization is a complex process during which bacteria combine with certain serum proteins and are rendered more susceptible to phagocytosis (19). Thus, any serum protein that fulfills this function may be termed as opsonin (3). Both IgG and IgM antibodies have significant opsonizing activities against gram-negative bacteria in the presence of heat-labile opsonins (18). Furthermore, IgG antibodies in convalescent sera have been shown to augment phagocytosis of P. aeruginosa even in the absence of heat-labile opsonins (3). The nature of heat-labile factors that promote phagocytosis has not been clearly defined (15). Available evidence, however, indicates that sequential reaction of Cl, C4, and C2, leading to the cleavage of C3 with resultant fixation of C3b on the bacterial surface, is the principal process of opsonization of pneumococci (11) and of P. aeruginosa (20). In the present study, all strains of P. aeruginosa recovered from sputum were shown to be coated with specific antibodies of IgG, IgA, and IgM classes and also with complement component C3. The state of coating in patients with
y-Chain -
a-Chain
,-Chain
-
-
+++ ++++
++++ ++++
+++ +++
+ + +++ ++++
+ + ++++ ++++
+ + +++ +++
CF does not seem to be different from those with chronic pulmonary infections of another nature, although the number of patients in the latter group was very limited. The fact that subcultured Pseudomonas, which was initially shown to be free of antibody and complement coatings, developed positive fluorescence after incubation with sputum eluates suggests that the coating process can take place in the lumen ofthe lower respiratory tract. Another possible site of coating would be the mucosa and submucosa of the respiratory tract. Here the invading pathogens will be acted upon by the humoral factors present in the tissue and then expelled into the sputum during the process of tissue necrosis, which is so often seen in patients with CF. It is reassuring to find that the sputum eluates contain significant levels of IgG, IgA, IgM, and C3. The exact process by which these humoral factors appear in the sputum eluates is not certain. It is possible that these humoral factors leaked out by bleeding through the damaged mucosal linings of the lower respiratory tract. Other possible mechanisms would be an inflammatory exudation, passive transudation, active transport of IgA (16), or a combination of these. One may well speculate that other components of complement may be present in the sputum, which, if is indeed the case, would suggest that complement-mediated bacteriolysis can take place in sputum in situ. Although the coating with IgA antibody was most intense, the role of IgA in promoting phagocytosis is not completely understood. IgA antibodies do not ordinarily fix complement (10). The aggregated IgA, however, can utilize the alternate pathway of complement activation and cleave C3 directly, resulting in fixation of C3b on the bacterial surface (4). Thus, our findings suggest that these organisms are opsonized in sputum and are potentially subject to enhanced phagocytosis by phagocytes. In support of this, a number of granulocytes and mononuclear cells in sputum were found to contain gram-negative rods, presumably Pseu-
VOL. 14, 1976
domonas, in the cytoplasm. In spite of these findings, local colonization and multiplication of P. aeruginosa continue in the lower respiratory tract of patients with CF. The extent and efficiency of the opsonization and phagocytosis is a matter of conjecture, since chemical and physical alterations of the local environment have been shown to affect these processes rather profoundly (5, 6). Apparently, requirements for phagocytosis by pulmonary alveolar macrophages are different from those for polymorphonuclear leukocytes (13). In addition, inherent functional alterations of the lower respiratory tract in CF, including abnormal mucociliary clearance, might not be favorable for the effective mobilization of, and phagocytosis by, phagocytes thus perpetuating Pseudomonas infections in the lung. ACKNOWLEDGMENTS This work was supported by grants from the Cystic Fibrosis Research Fund, the Cystic Fibrosis Foundation, the Philadelphia Pediatric Pulmonary Disease Program, and a Pulmonary Academic Award from the National Heart and Lung Institute (D.S.H.). 1. 2.
3.
4.
5.
6.
LITERATURE CITED Biggar, W. D., B. Holmes, and R. A. Good. 1971. Opsonic defect in patients with cystic fibrosis of the pancreas. Proc. Natl. Acad. Sci. U.S.A. 68:1716-1719. Boxerbaum, B., M. Kagumba, and L. W. Matthews. 1973. Selective inhibition of phagocytic activity of rabbit alveolar macrophages by cystic fibrosis serum. Am. Rev. Respir. Dis. 108:777-783. Boyden, S. V., R. J. North, and S. M. Faulkner. 1976. Complement and activity of phagocytes, p. 190. In G. E. W. Wolstenholme and J. Knight (ed.), Ciba Foundation on complement. Little Brown, and Co., Boston. Gotze, O., and H. J. Muller-Eberhard. 1971. The C3activator system: an alternative pathway of complement activation. J. Exp. Med. 134:90s-108s. Green, G. M., and D. Carolin. 1967. The depressant effect of cigarette smoke on the in vitro antibacterial activity on alveolar macrophages. N. Engl. J. Med. 276:421-427. Green, G. M., and E. H. Kass. 1964. Factors influencing
P. AERUGINOSA Ig AND C IN SPUTUM
7.
8.
9. 10.
11.
12.
13. 14.
15.
16.
17.
18.
19.
20.
117
the clearance of bacteria by the lung. J. Clin. Invest. 43:769-776. Gugler, E., J. C. Pallavicini, H. Swerdlow, I. Zipkin, and P. A. di Sant'Agnese. 1968. Immunological studies of submaxillary saliva from patients with cystic fibrosis. J. Pediatr. 73:548-559. Huang, N. N., E. L. Van Loon, and K. T. Sheng. 1961. The flora of the respiratory tract of patients with cystic fibrosis of the pancreas. J. Pediatr. 59:512-521. Iacocca, V. F., M. S. Sibinga, and G. J. Barbero. 1963. Respiratory tract bacteriology in cystic fibrosis. Am. J. Dis. Child. 106:315-324. Ishizaka, T., K. Ishizaka, T. Boros, and H. Rapp. 1966. C'l fixation by human isoagglutinins: fixation of Cz by yG and yM but not by yA antibody. J. Immunol. 97:716-726. Johnston, R. B., Jr., M. R. Klemperer, C. A. Alper, and F. S. Rosen. 1969. The enhancement of bacterial phagocytosis by serum. The role of complement components and two cofactors. J. Exp. Med. 129: 1275-1290. Marcer, V., G. Mastella, and V. Mengoli. 1970. Immunoglobuline nel sierro, nell'escreato e nella saliva di pazienti affetti da fibrosi cistica (mucoviscidosi) con importanti complianze respiratorie. Riv. Clin. Pediatr. 83:135-149. Oren, R., A. E. Farnham, K. Saito, E. Milofsky, and M. Karnovosky. 1963. Metabolic patterns in three types of phagocytizing cells. J. Cell. Biol. 17:486-501. Reynolds, H. Y. 1974. Pseudomonas in cystic fibrosis, p. 7. In GAP conference report, June 13, 1974. National Cystic Fibrosis Research Foundation, Atlanta, Ga. Smith, M. R., H. S. Shin, and W. B. Wood. 1969. Natural immunity to bacterial infections. The relation of complement to heat-labile opsonins. Proc. Natl. Acad. Sci. U.S.A. 63:1151-1156. South, M. A., W. J. Warwick, F. A. Wollheim, and R. A. Good. 1967. The IgA system. III. IgA levels in the serum and saliva of pediatric patients - evidence for a local immunological system. J. Pediatr. 71:645-653. Thomas, V., A. Shelokov, and M. Forland. 1974. Antibody-coated bacteria in the urine and the site of urinary tract infection. N. Engl. J. Med. 290:588-590. Williams, R. C., Jr., J. H. Dosett, and P. G. Quie. 1969. Comparative studies of immunoglobulin opsonins in osteomyelities and other established infections. Immunology 17:249-265. Winkelstein, J. A. 1973. Opsonins: their function, identity and clinical significance. J. Pediatr. 82:747-753. Young, L. S., and D. Armstrong. 1972. Human immunity to Pseudomonas aeruginosa. I. In-vitro interaction of bacteria, polymorphonuclear leukocytes, and serum factors. J. Infect. Dis. 126:257-276.
