Eur Arch Otorhinolaryngol (1992) 249 : 374-379

European Archives of

Oto-RhinoLaryngology © Springer-Verlag 1992

Pneumococcal antigens and serum antibody responses in experimentally induced sinusitis K. M. Westrin, A. Freijd, and P. Stierna Department of Otorhinolaryngology, Huddinge Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden Received January 16, 1992 / Accepted June 16, 1992

Summary. Maxillary sinusitis was induced in New Zealand White rabbits with Streptococcus pneumoniae serotype 3 and the serum antibody response was recorded by enzyme immunoassay. Activity of the three major immunoglobulin classes, viz. IgG, IgA and IgM, against the type-specific capsular polysaccharide, the cell wall Cpolysaccharide as well as its subunit phosphorylcholine was analyzed. A pronounced increase in immunoglobulins reactive to the specific capsular antigen was observed throughout the study period, with the rise being particularly noticeable during the first 2 weeks. An increase in anti-C-polysaccharide antibodies was also evident, but no reaction to phosphorylcholine could be detected. Although the histological findings at 3 and 4 weeks varied in spite of similar serum antibody responses, a correlation appeared to exist between a more rapidly increasing anti-capsular IgG production and the prevalence of a purulent sinus secretion. Key words: Sinusitis - Streptococcus pneumoniae - Bacterial capsule - Cell wall - Antibodies

coccal cell wall [16] and its major antigenic determinant, phosphorylcholine (PC) [10]. High concentrations of antibodies to CPS in both serum and secretion seem to lack a significant protective effect against pneumococcal infection in man [14, 19, 27]. However, anti-PC antibodies do protect mice from experimentally induced pneumococcal infections [4, 35]. In the rabbit, sinusitis induced with S.pneumoniae serotype 3 is a self-limiting infection, and pneumococci can only rarely be found in the sinus cavity more than 1 week after induction [34]. It was therefore of interest to us to investigate whether sinusitis induces a serum antibody response to the infecting microorganism, and whether any relationship exists between specific humoral reactions and the degree of mucosal inflammation present. For this purpose, an enzyme immunoassay (EIA) was used, analyzing antibodies of the three major Ig classes directed against the three pneumococcal antigens, capsular polysaccharide of serotype 3 (PPS3), CPS and PC. An attempt was also made to determine whether development of antibodies to PC in this model could be used to monitor individual responsiveness to polysaccharide antigens [5].

Introduction Materials and methods

Streptococcus pneurnoniae is a major pathogen in acute sinusitis in man, constituting roughly half of all microbial isolates from purulent antral aspirations in untreated patients [3, 7]. The capsular polysaccharide is regarded as the most significant virulence factor of this microorganism. The capsule lacks toxicity of its own, but inhibits phagocytosis [1]. In clinical studies aiming to evaluate the immunologic response to pneumococcal infection, nasopharaynegal carriage or vaccination, antibodies to the various type-specific capsular polysaccharides (PPS) have been analyzed [2, 6], as well as antibodies to the C- polysaccharide (CPS), a major constituent of the pneumo-

Correspondence to: K. M. Westrin

S. pneumoniae serotype 3 was dissolved in saline to a concentration of approximately 108 cfu/ml. Unilateral sinusitis was induced in 16 inbreeding-free New Zealand White rabbits by injecting 1 ml of the bacterial suspension into an occluded sinus cavity [15]. Prior to inoculation, and weekly thereafter (at 1, 2, 3, 4, 8 and 12 weeks), blood samples were drawn from the auricular vein, and serum was stored at -70°C until analysis. Visual assessment of mucosal edema and sinus secretions was made at the time that the animals were sacrificed. Bacterial cultures were also taken from the sinus cavity at this time.

Histological techniques For histological evaluation, mucosal samples were prepared by perfusion-fixation with 3% glutaraldehyde in 0.1M sodium caco-

375 Table 1. Median serum antibody activity of the three major isotypes against PPS3, CPS and PC, expressed as optical density at 492 nm in enzyme immunoassay at differrent time intervals of experimental pneumococcal sinusitis

CPS

PC

Week Animals

PPS3

(n)

IgG

IgA

IgM

IgG

IgA

IgM

IgG

IgA

IgM

0 1 2 3 4

16 16 16 16 10

0.21 0.80 1.52 1.54 2.00

0.30 0.43 0.47 0.60 0.55

0.25 0.42 0.36 0.31 0.26

0.24 0.27 0.41 0.44 0.61

0.33 0.36 0.36 0.38 0.45

0.25 0.28 0.28 0.27 0.27

0.21 0.23 0.29 0.25 0.29

0.21 0.22 0.24 0.28 0.25

0.21 0.20 0.20 0.22 0.20

dylate buffer and post-fixation with 1% O s O 4 in the same buffer. After dehydration in a graded series of ethanol, the specimens were embedded in Epon 812. Tissue sections ( l g m thickness) were stained with toluidine blue for light microscopy, and ultrathin (70 nm) sections were collected on copper grids and contrasted with uranyl acetate and lead citrate for transmission electron microscopy. Encapsulation of the pneumococci was assessed before injection and in antral aspirates obtained 1 and 2 days after initiating infection. After fixation, smears were stained with crystal violet, safranine and India ink for capsule visualization under a light microscope. The area occupied by single bacterial cells was measured with a Macintosh Image 1000 connected to Scion Image Capture 2 and Video Frame Grabber Card.

3 eq

•

d

j

2

Antigens PPS3 was obtained from Merck, Sharp and Dohme (Rahway, N. J., USA), CPS was donated by AnnMargret Sj6gren, Department of Bacteriology, Karolinska Institute, Stockholm. Diazophenyl-phosphorylcholine was synthesized from nitrophenyl-phosphorylcholine (Sigma, St. Louis, Mo., USA) by courtesy of Anna-Karin Tid6n, Department of Organic Chemistry, Stockholm University [8], and coupled to rabbit serum albumin (Sigma).

0

1

t

I

4

8

12

Week Fig.1. The appearance of IgG production against PPS3 (O), CPS (O) and PC (D), expressed as mean optical density (OD) values +_ SD in enzyme immunoassay during 12 weeks (n = 16 at 1, 2, 3 weeks; n = 10 at 4 weeks; n = 4 at 8 weeks; and n = 2 at 12 weeks)

Enzyme immunoassay Results Microtiter plates (Costar Corp., Cambridge, Mass., USA) were coated overnight with either PPS 3 (10 gg/ml) in phosphate buffer, CPS (20 gg/ml), or PC (20 gg/ml) in the same buffer. The plates were then incubated with serum samples in different dilutions (10-2-10 -3) for 4 h at room temperature. Between each step, the plates were washed three times in saline with Tween added. The plates were incubated overnight at room temperature with horseradish peroxidase conjugated goat antibodies directed against rabbit IgA (diluted 1 : 4000) (Nordic Immunology, Tilburg, The Netherlands), IgG (1 : 6000) or IgM (1:4000) (Southern Biotechnology, Birmingham, Ala., USA). Development was performed with 1, 2phenylenediamine dihydrochloride (OPD) chromogen substrate (Dakopatts, Copenhagen, Denmark). The reaction was allowed to continue for 15 min (PPS3), 30 min (CPS) or 45 min (PC) and absorbance was then read at 492nm. In order to evaluate the significance of any cross-reaction or contamination with CPS in the PPS3 antigen [26], anti-CPS antibodies were excluded by adding an excess amount of CPS (50 gg/ml) to serum samples before measuring the PPS3 antibodies [16].

Statistical analysis The non-parametric methods used for analysis of OD values were Wilcoxon's paired signed rank test and the Mann-Whitney U-test. Differences incapsular size were tested with Student's t-test (twotailed).

Anti-PPS 3 antibodies I n c r e a s e d I g G r e a c t i v i t y to PPS3 was s e e n in all a n i m a l s d u r i n g t h e first 2 w e e k s after i n f e c t i o n , with t h e levels rem a i n i n g high t h r o u g h o u t t h e p e r i o d s t u d i e d (Fig. 1). I g A activity against PPS3 was i n c r e a s e d d u r i n g the first 3 w e e k s ( T a b l e 1). A n i n c r e a s e in I g M was s e e n d u r i n g t h e 1st w e e k a n d was f o l l o w e d b y a d e c r e a s e f r o m t h e 2 n d w e e k o n w a r d ( T a b l e 1).

Anti-CPS antibodies I n all a n i m a l s a rise in a n t i - C P S I g G activity was elicited, b u t t h e i n t e n s i t y o f this c h a n g e v a r i e d i n d i v i d u a l l y . T h e i n c r e a s e was significant d u r i n g t h e first 2 w e e k s a f t e r infection, w i t h t h e activity r e m a i n i n g at e l e v a t e d levels t h r o u g h o u t t h e 12-week p e r i o d (Fig. 1). A slight i n c r e a s e in I g A activity was also s e e n , b u t this was significant o n l y a f t e r 3 w e e k s . N o c h a n g e c o u l d b e d e t e c t e d in I g M activity. When anti-CPS antibodies were excluded from the s e r u m s a m p l e s , t h e r e was n o significant c h a n g e in I g G activity against PPS3.

376 1600,

ever, no obvious relation existed between histologic features and immunologic response.

1400-

Capsule measurements

1200 0 0 0

I000 I

0

The capsular stain revealed an increased capsule size in microorganisms growing in the rabbit sinus cavity (Fig. 5). The average area occupied by the 25 bacteria with largest capsule/field was 8.4 ~tm2+ 2.6/cell after 2 days in the sinus cavity, compared with 5.7 gm2+ 1.8 before inoculation (P < 0.001).

800 0 0

O

600

O 400

200

0

0 0 0

O

0 Fig. 2. Increase in IgG activity against PPS3 for each rabbit after 3 weeks in animals with sinus purulence (right) and without purulence (left). The percentage increase in OD between 0 and 3 weeks has been calculated (P < 0.05). (Histological preparations of mucosal samples from rabbits marked with asterisk are depicted in Figs. 3, 4)

Anti-PC antibodies As shown in Table 1, no antibody response could be recorded against PC.

Degree of inflammation A considerable amount of purulent secretion was found in eight rabbits, while four animals had serous secretions only. No secretions were present in the remaining four animals. When comparing the purulent and non-purulent groups serologically, a more rapid increase in IgG against PPS3 was seen in the purulent group (Fig. 2). However, the long-term absorbance values for IgG did not differ with respect to the character of the secretions. The light- and electron-microscopic studies revealed a varying degree of edema, inflammatory cell infiltration, regeneration of goblet cells, and occasional ciliary damage of the epithelial cells lining the mucosa. Purulence in the sinus could histologically be reflected by increased thickness of the epithelium as well as increased numbers of goblet cells. The mucosal architecture and ultrastructure of an animal with sinus purulence and a rapid serologic response is illustrated in Fig. 3. For comparison, the picture of a "slow responder" with serous secretions in the sinus cavity is shown in Fig. 4. How-

Discussion Due to its ability to prevent phagocytosis, the pneumococcal capsule is very important in the establishment of clinical infections. In the rabbit, Tuomanen et al. [33] demonstrated the role of the serotype 3 capsule in causing experimentally induced pulmonary inflammation. The capsular antigens have also been shown to induce protective antibody production in man as well as in several species of experimental animals (e.g. [2, 9, 17, 24]). The most evident E I A antibody response in our present animal model was directed towards PPS3 and IgG showed the most significant increase in activity of all the three major isotypes studied. The strong response to PPS3 thus concurs with previous reports, i.e., infection or vaccination reliably elicits a specific antibody production against the type 3 capsule [16]. In experimentally induced sinusitis an anti-capsular IgG production is recorded after 1 week, with sustained high serum antibody levels still present after 12 weeks. In otitis media induced in rats, the anti-PPS3 IgG response also appeared with 4-7 days, but was followed by a rather rapid decline in serum levels [29]. An IgG response to CPS was evident in all rabbits. Since exclusion of anti-CPS antibodies did not modify the activity of IgG against PPS3, the purity of PPS3 was confirmed and the antibodies to it were considered typespecific. The weaker respone - as well as the slight time lag - seen in IgG development against CPS vis-a-vis PPS3 may be related to an initial masking of cell wall determinants by the capsular polysaccharide [28, 35]. A substantial amount of CPS would no doubt be expected to be released to the environment by bacterial autolysis [18] with a subsequent antibody production in the host [25]. The rapid increase in anti-PPS3 IgG recorded indicates that bacteria had already been phagocytosed to a large extent before the onset of massive autolysis, so that the immunization process to CPS was consequently blocked. Rabbits may be unable to synthesize Ig against PC, as it has been suggested by Szu et al. [30] that rabbit antibodies to CPS are directed against the saccaride "backbone" and not against PC. However, most likely an antiPC Ig actually exists in the rabbit (personal communication, Dr. A. M. SjOgren). PC is not an exclusive subunit of pneumococcal CPS, but can be isolated from other streptococci, lactobacillus, certain nematodes and fungal species [21]. Whether these antibodies occur naturally in

377

Fig. 3a-e. Morphologic features of animals found to be serologic rapid responders. a Light micrograph displaying increased thickness of the epithelial layer, localized losses of cilia, infiltration of inflammatory cells and depletion of serous granules in subepithelial glands. Bar = 25 gin; toluidine blue, × 390. b Transmission electron microscopy (TEM) of the same specimen. Goblet cells contain mucous granules of varyiny density. Bar = 5gin, x 2500. c Electron micrograph showing inflammatory cells penetrating the mucosal epithelium. The cilia are irregularly shaped and reduced in number. Nuclei of the cylindrical cells are located at different levels. × 3000

the rabbit, as in mice, is at present not clearly understood. The lack of an immunologic reaction to PC in our experiment is suggestive of a natural occurrence in the rabbit, and measurement of anti-PC Ig synthesis does not seem to be much use as an indicator in this species for its ability to produce antibody upon immunization with polysaccharide antigens. Pneumococcal cell-wall-associated antigens are of great importance and contribute to bacterial toxicity [22]. In the chinchilla middle ear, isolated pneumococcal cell wall induces epithelial metaplasia, subepithelial congestion, granulation tissue formation and an infiltration of polymorphonuclear leukocytes [22], similar to the short-term inflammatory findings in experimentally induced sinusitis [34]. In otitis media induced by pneumococcal cell wall components in the guinea pig, inflammatory cell influx and vascular permeability to the middle ear can be demonstrated as early as after 2-6 h [20]. In the lungs, an inflammatory response in bronchoalveolar lavage fluid can be recorded 4 h after intratracheal challenges with purified cell wall components [33], and similar observations have been recorded in the meninges [31, 32]. In our present model, S. p n e u m o n i a e can only rarely be isolated from the sinus cavity after more than 1 week, but bacterial multipli-

cation does not always seem to be a prerequisite for inflammation. It has also been shown that bacterial lytic products such as cell wall components released during autolysis may constitute a reservoir of inflammatory products that may contribute to further tissue injury [33]. Animal passage of pneumococci enhances organism virulence [15]. The passage is believed to stimulate capsule synthesis and/or select more encapsulated bacteria for growth. A reduction in or loss of capsular substance can be the result of suboptimal substrate conditions, such as obtained when culturing organisms in vitro [28]. In the antral aspirate smears from our animals, pneumococci with prominent capsule appeared to increase in number during the first 2 days after inoculation. It is possible that the demonstrated increase in capsular size may be due to a secondary response of secretory antibodies [23, 27], provoking a "Quellung reaction." However, this could only occur if the rabbits had been immunized previously to substances cross-reacting with PPS3, with seems unlikely. Increasing encapsulation and sustained high anti-capsular IgG levels imply a greater importance of bacterial growth (stimulating production of anti-capsular antibody) and diminished importance of cell wall toxin release per

378 se (enhanced by a low leukocyte phagocytotic activity). In comparison, with respect to its short duration, the otitis media induced by S.pneumoniae serotype 3 in the rat [12] may be considered to represent a model with a m o r e acute toxin-induced inflammation. The anti-capsular I g G m a x i m u m in that model was already reached within i week [29], and gross and microscopic findings in the ear were completely normalized within 9 days without treatment [12]. Nevertheless, the prophylactic effect mediated by antibiotic treatment (penicillin V) in the rat middle ear [13] suggests the importance of viable bacteria for the induction of inflammation in this animal model. In general, a m o r e p r o n o u n c e d tissue inflammation can be expected when local I g G predominates over local I g A [11]. In experimentally induced sinusitis in the rabbit, rapidly increasing anti-capsular I g G production could be correlated with the occurrence of purulent secretions. In pneumococcal sinusitis a production of circulating antibodies is p r o v o k e d and is mainly of the I g G class. These antibodies are directed against capsular and, to a lesser degree, cell wall polysaccharide antigens. In general, a m o r e rapid anti-capsular I g G response was seen in rabbits, with a stronger inflammatory response in the mucosa and with purulent secretions in the lumen. However, the histologic picture could vary somewhat among animals, irrespective of I g G levels. This suggests that the individual rate of the humoral Ig response corresponds m o r e closely to the severity of the infection than to its resolution. The generation of an inflammatory response in the course of a natural infection serves to control bac-

Fig.4a, b. Morphologic features of a serological slow responder. a Survey view of mucosa with light microscopy. The epithelial layer is of normal thickness and the nuclei of ciliated cells are approximately at the same level. Numerous dark granules are seen in the luminal part of each glandular serous cell. Toluidine blue, × 470. b Detail of same mucosa using TEM. Note the regular size and shape of the cilia. Basal cells are situated between the basal membrane and columnar cells. Dark granulated cells are scattered between the ciliated cells. × 3200

Fig. 5a, b. Visualization of the pneumococcal capsules using crystal violet-safranine-India ink staining of smears from a bacterial cultures prior to inoculation, and b sinus aspirates of pus 2 days after inoculation. The capsule appears as a pale halo around each bacterial cell. Note the higher incidence of a more distinct capsule in b. The differing capsular size observed within populations is caused to some extent by the different levels of focus x 1000

379 terial m u l t i p l i c a t i o n , b u t the histologic r e a c t i o n d e p i c t e d is n o t i n v a r i a b l y reflected in a given serologic r e s p o n s e , as seen in the r a b b i t sinus m u c o s a .

Acknowledgements. The study was supported by grants from the Swedish Medical Research Council (project no. 00749), the Swedish Society for the Medical Sciences, the Swedish Society of Medicine, the Torsten and Ragnar S6derberg Foundation and the Swedish Otolaryngological Society. The technical assistance of Ms. A. Jonsson and Dr. A. Samuelsson is gratefully appreciated. Dr. A. M. Sj6gren and Dr. B. Carls66 are thanked for their constructive discussions.

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