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* Laboratory for Experimental Internal Medicine, Departments of
Pathology and
Nuclear Medicine, and
Laboratory for Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit is rejoined by a noncovalent linkage with the TM7/cytoplasmic 
subunit. Due to alternative RNA splicing, the number of EGF domains in EGF-TM7 receptors is variable (8). For human CD97 (hCD97), isoforms with three, four, and five EGF domains have been identified (4), designated hereafter as hCD97(EGF1,2,5), hCD97(EGF1,2,3,5), and hCD97(EGF1,2,3,4,5). Mouse CD97 (mCD97) also exists in three isoforms (5, 6). Next to isoforms with three and four EGF domains, indicated as mCD97(EGF1,2,4) and mCD97(EGF1,2,3,4), a third isoform mCD97(EGF1,2,X,3,4) was detected. This isoform has a sequence of 45 aa between the second and third EGF domain that does not correspond to known protein modules. EGF domains 3 and 4 in mCD97 are the homologues of EGF domains 4 and 5 in hCD97, respectively (6). Schematic structures of CD97 in humans and mice are depicted in Fig. 1.
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We previously identified decay accelerating factor (CD55) as a cellular ligand of CD97 (30). CD55 is a GPI-linked molecule that prevents complement deposition on self cells by inhibiting C3/C5 convertases (31, 32). The binding site for CD55 is formed by the EGF domain region (33). Lin et al. (34) showed that hCD97(EGF1,2,5) binds CD55 with low affinity (86 µM) and a rapid off-rate (0.6 s-1). Affinity for CD55 differs between CD97 isoforms and is significantly lower for the larger isoforms hCD97(EGF1,2,3,5), hCD97(EGF1,2,3,4,5), and mCD97(EGF1,2,X,3,4) (6, 33, 34). Efficient up-regulation of CD97 and CD55 under physiological and pathological conditions (1, 7, 24, 35, 36) implies that generation of cellular interactions of high avidity in vivo is regulated at the expression level of both molecules (37). More recently, chondroitin sulfate has been identified as a second ligand of CD97 (38). This cell surface glycosaminoglycan (GAG) side chain specifically interacts with the larger isoforms of CD97 (for details see Discussion).
Based on the unusual structure of the subunit, the ability of this region to bind cellular ligands, and the rapid up-regulation of CD97 on activated lymphocytes, a role of CD97 in cell adhesion and migration has been suggested (2, 4, 7, 30, 39). Supportive evidence for this assumption was recently received from studies in intestinal carcinomas. Aust and coworkers (28, 29) showed, first, that expression levels of CD97 correlate with the in vitro migration and invasion capacity of colorectal tumor cell lines, second, that migration and invasiveness of the fibrosarcoma cell line HT-1080 can be increased 2- to 3-fold by inducing (tetracycline-off control) expression of CD97, and, third, that scattered tumor cells at the invasion front of colorectal and gastric carcinomas are stronger CD97 positive than tumor cells in solid formations of the same tumor.
In this study, we report for the first time in vivo data on CD97. In two murine models we investigated the effect of newly generated mCD97 mAb on the migration of neutrophilic granulocytes. First, in experimental colitis, mCD97 mAb caused a delay in the homing of adoptively transferred neutrophils to the colon. Second, in mice intranasally (i.n.) inoculated with Streptococcus pneumoniae, application of mCD97 mAb impaired the recruitment of neutrophils to the lungs thereby reducing resistance to pneumonia. Together, these data are in favor of an essential role of CD97 in leukocyte migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Novel mCD97 mAb were generated in Armenian hamsters (Cricetulus migratorius; Cytogen, West Roxbury, MA) as described recently (6). In short, the Armenian hamster fibroblast line ARHO12 was transfected with mCD97(EGF1,2,3,4) cDNA in pcDNA3.1/Zeo(+) (6). Cells were selected with zeocin (Invitrogen, Leek, The Netherlands) at 500 mg/ml in culture medium. Resulting stably transfected clones were tested for mCD97 expression by flow cytometry with the earlier generated mCD97 mAb 1A2 (6). One clone was selected and used for immunization. Three i.p. injections with 10 x 106 irradiated (50 Gy) cells in PBS were given at weekly intervals. Eight weeks after the third injection, the hamster was boosted i.p. with 10 x 106 cells. Three days later, hamster spleen cells were fused with mouse myeloma SP2/0 cells by standard hybridoma technology. Binding of hybridoma supernatants to the ARHO12 clone stably expressing mCD97(EGF1,2,3,4) was tested by flow cytometry. Flow cytometry of COS cells expressing mCD97 isoforms or chimeras between mCD97 and hCD97 was used to identify hybridomas that recognize different regions of mCD97. Selected hybridomas were subcloned until they were monoclonal and stable. The hybridomas 1B2, 1C5, and 1D2 were grown at large amounts and Ig was purified using protein A-Sepharose CL-4B (Sigma-Aldrich, St. Louis, MO).
Generation of chimeric m-hCD97 constructs
Generation of an expression construct encoding mCD97(EGF1h,2,4) by overlap extension-PCR has been described earlier (6). In a similar manner, constructs in which EGF domain 1 or EGF domains 1 and 2 of mCD97 have been exchanged by homologues EGF domains of hCD97 were made. In a first step, two overlapping PCR fragments were generated in separate PCRs. For hCD97(EGF1m,2,5), the N-terminal part of mCD97 including EGF domain 1 was amplified from mCD97 cDNA (6) using a standard T7 primer ((+) strand) and the specific primer 5'-CGTTGATGTCTTCACAGCTCTCTGCAGGGTTAG-3' (nt 214–237 of the (-) strand; the sequence overlapping with EGF domain 2 of hCD97 is underlined). EGF domains 2 and 5 and part of the stalk region of hCD97 were amplified from hCD97 cDNA (2) using the specific primers 5'-GCTGTGAAGACATCAACGAGTGTGCAACAC-3' (nt 261–281 of the (+) strand; the sequence overlapping with EGF domain 1 of mCD97 is underlined) and 5'-GGAATGCAGGTTCAAGGAGGC-3' (nt 1031–1051 of the (-) strand). For hCD97(EGF1m,2m,5), the N-terminal part of mCD97 including EGF domains 1 and 2 was amplified using a standard T7 primer ((+) strand) and the specific primer 5'-CGTCCACATCTTGACATGTATTCTCACTCTCG-3' (nt 368–390 of the (-) strand; the sequence overlapping with EGF domain 5 of hCD97 is underlined). EGF domain 5 and part of the stalk region of hCD97 were amplified using the specific primers 5'-CATGTCAAGATGTGGACGAGTGCAGCTCCGG-3' (nt 417–438 of the (+) strand; the sequence overlapping with EGF domain 2 of mCD97 is underlined) and 5'-GGAATGCAGGTTCAAGGAGGC-3' (nt 1031–1051 of the (-) strand). The PCR fragments were purified and mixed together to serve as template for the second step of the overlap extension-PCR performed for both constructs with a standard T7 primer and the specific primer 5'-GGAATGCAGGTTCAAGGAGGC-3'. Chimeric constructs were obtained by replacing the N-terminal part of hCD97(EGF1,2,5) cDNA in pcDNA3.1/Zeo(+) with the PCR amplicons, thereby using a HindIII site in the multiple-cloning site of the vector and a EcoRV site in the hCD97 sequence.
To generate a mCD97(EGF1h,2h,4) construct, the N-terminal part of mCD97 including EGF domain 1 and part of EGF domain 2 was amplified using a standard T7 primer ((+) strand) and the specific primer 5'-CTTGAATGTTTTTGCCCCAGAAAC-3' (nt 368–391 of the (-) strand). A chimeric construct was obtained by replacing the N-terminal part of mCD97(EGF1,2,4) cDNA in pcDNA3.1/Zeo(+) with the PCR amplicon, thereby using a NheI site in the multiple-cloning site of the vector and a HpaI site in the mCD97 sequence (blunt-end ligation of the 3' end of the amplicon).
COS cell expression and mapping of mAb binding sites
COS cells were transfected with mCD97, hCD97, or chimeric m-hCD97 constructs using Lipofectamine Plus reagent (Life Technologies, Gaithersburg, MD). Three days after transfection, reactivity of mCD97 mAb 1A2, 1B2, 1C5, and 1D2 was analyzed by standard flow cytometry on a FACScan (BD Biosciences, Mountain View, CA). Control stainings were performed with hCD97 mAb CLB-CD97/1 and CLB-CD97/3 (40). PE-conjugated goat anti-hamster Ig (Southern Biotechnology Associates, Birmingham, AL) or PE-conjugated goat anti-mouse Ig (Immunotech, Marseille, France) were used as second step reagent.
Erythrocyte adhesion studies
Adhesion studies were performed as described previously (6, 30, 33) by overlaying COS cells in 12-well cell culture plates three days after transfection with 50 x 106 mouse erythrocytes for 30 min at room temperature. Nonadhering cells were removed by gentle washing with PBS before examination by microscopy. To test for blocking capacity, mCD97 mAb were added to the erythrocyte suspension at a final concentration 5 µg/ml.
Induction of dextran sulfate sodium (DSS) colitis
Pathogen-free 8-wk-old female BALB/c mice were obtained from Harlan (Zeist, The Netherlands). The Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands) approved all experiments described in this manuscript. To induce DSS colitis, mice were fed 4% (w/v) DSS (TdB Consultancy, Uppsala, Sweden) in their drinking water for at least 7 days (41).
Adoptive transfer of technetium-99m (99mTc)-labeled neutrophils and pinhole single photon emission computed tomography (SPECT)
Peritoneal granulocytes were harvested by rinsing the peritoneal cavity with 5 ml of sterile PBS 5 h after i.p. injection of 1 ml of 10% proteose peptone (Difco, Detroit, MI) in PBS. A total of 5–10 x 106 cells/mice were harvested and labeled with 75 MBq of freshly prepared 99mTc-hexamethylpropylene amine oxime (HMPAO) according to manufacturers’ guidelines (Ceretec; Amersham Health, Eindhoven, The Netherlands). After 15 min, cells were washed and resuspended in saline, containing 5 µg/ml Ab. Next to mCD97 mAb 1B2, hamster Ig (Rockland, Gilbertsville, PA) was used as control (cIg).
Within 30 min after labeling, 
5 x 106 cells, labeled with 40 MBq 99mTc-HMPAO, were injected i.v. in a tail vein of mice with DSS colitis (n = 7–8). One hour later, scintigraphy was performed on rotating mice for three-dimensional imaging using a recently described and validated high-resolution pinhole SPECT technique (42). During the whole procedure, animals were sedated with fentanyl and fluanisone (Janssen Pharmaceutica, Beerse, Belgium) and diazepam (Roche, Mijdrecht, The Netherlands). SPECT reconstruction was done using a HERMES (Nuclear Diagnostics, Stockholm, Sweden) application program, using filtered back projection adapted to pinhole SPECT.
To determine the radioactivity uptake semiquantitatively, and to correct for differences in total amount of radioactivity administered and variable numbers labeled granulocytes, the colon uptake ratio was determined as follows. Five consecutive transverse slices with the highest colon uptake were selected and added. Regions of interest were set for the colon and abdominal background (not containing the colon) and the number of counts in each region of interest was measured. The colon uptake ratio was calculated by subtracting background activity from the colon activity and subsequently dividing the corrected colon uptake by the background to yield a specific uptake ratio: (counts colon-counts background)/(counts background).
Induction of pneumonia
Pathogen-free 8-wk-old female BALB/c mice were obtained from Harlan Sprague Dawley. Pneumonia was induced as described previously (43, 44, 45). Briefly, S. pneumoniae serotype 3 was obtained from American Type Culture Collection (ATCC 6303; Rockville, MD). Pneumococci were grown for 6 h to mid-logarithmic phase at 37°C in 5% CO2 using Todd-Hewitt broth (Difco), harvested by centrifugation at 1500 x g for 15 min, and washed twice in sterile isotonic saline. Bacteria were then resuspended in sterile isotonic saline at a concentration of 1 x 104 CFU/50 µl, as determined by plating serial 10-fold dilutions on sheep-blood agar plates. Mice were lightly anesthesized by inhalation of isoflurane (Upjohn, Ede, The Netherlands) and 50 µl were inoculated i.n.
Abs 1B2, 1C5, and cIg were given i.p. at doses of 0.5 mg 24 h before, 24 and 72 h after induction of pneumonia. The size of the groups was n = 10 for the survival study and n = 8 for the remaining assays.
Preparation of lung homogenates and determination of bacterial outgrowth
At 20 and 44 h after inoculation, mice were anesthetized with hypnorm (Janssen Pharmaceutica) and midazolam (Roche) and sacrificed by bleeding out the vena cava inferior. Blood was collected in EDTA-containing tubes. Whole lungs were harvested and homogenized at 4°C in four volumes of sterile saline using a tissue homogenizer (BioSpec Products, Bartlesville, OK). CFU were determined from serial dilutions of lung homogenates and blood, plated on blood agar plates and incubated at 37°C at 5% CO2 for 16 h before colonies were counted.
Histologic examination of lungs
Lungs for histologic examination were harvested at 20 h and 44 h after inoculation, fixed in 10% formalin and embedded in paraffin. Four-micrometer sections were stained with H&E and analyzed by a pathologist who was blinded for groups. For granulocyte staining (46), slides were deparaffinized and endogenous peroxidase activity was quenched by a solution of methanol/0.03% H2O2. After digestion with a solution of pepsine 0.25% in 0.01 M HCl, the sections were incubated in 10% normal goat serum and then exposed to FITC-labeled mouse Ly-6G mAb (BD PharMingen, San Diego, CA). Slides were incubated with a rabbit anti-FITC Ab (DAKO, Glostrup, Denmark) followed by a further incubation with a biotinylated swine anti-rabbit Ab (DAKO), rinsed again, incubated in a streptavidin-ABC solution (DAKO) and developed using 1% H2O2 and 3.3'-diaminobenzidin-tetra-hydrochloride in Tris-HCl. The sections were mounted in glycerin gelatin with a light methylgreen counter staining and analyzed.
Cytokine and chemokine determinations
For cytokine and chemokine measurements, lung homogenates were diluted 1 to 2 in lysis buffer containing 300 mM NaCl, 30 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 1% Triton X-100, and pepstatin A, leupeptin, and aprotinin (all 20 ng/ml; pH 7.4) and incubated at 4°C for 30 min. Homogenates were centrifuged at 1500 x g at 4°C for 15 min, and supernatants were stored at -20°C until assays were performed. Cytokines (TNF-, IL-1) and chemokines (KC, macrophage-inflammatory protein-2 (MIP-2)) were measured using specific ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.
Blood cell differentiation
EDTA blood was collected from mice 24 h after i.p. administration of 0.5 mg 1B2 or cIg per mouse (n = 4). Whole blood counts were determined and the number of neutrophils was calculated from these totals, using cytospin preparations stained with modified Giemsa stain (Diff-Quick; Baxter Diagnostics, McGraw Perk, IL).
Statistical analysis
Differences between groups were calculated by Mann-Whitney U test. For survival analyses, Kaplan-Meier analysis followed by log rank test was performed. Values are expressed as mean ± SEM. A two-tailed p value of < 0.05 was considered to represent a significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate mAb with different binding sites on the mCD97R, an Armenian hamster was immunized with ARHO12 cells stably expressing mCD97(EGF1,2,3,4). Screening of hybridoma supernatants for mCD97 specificity identified > 100 positive clones. mAb with disparate binding sites were detected when analyzing binding to COS cells expressing different mCD97 isoforms and m-hCD97 chimeras. The mAb 1B2, 1C5, and 1D2 were selected and analyzed in detail, together with the earlier generated mAb 1A2 (6), for binding specificity using an extended panel of m-hCD97 chimeras. As shown in Table I, 1B2 binds EGF domain 1, 1A2 binds EGF domain 2, and 1C5 and 1D2 bind EGF domain 3. Accordingly, 1C5 and 1D2 recognize mCD97(EGF1,2,3,4) and mCD97(EGF1,2,X,3,4) but not the smallest isoform mCD97(EGF1,2,4).
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Binding of CD97 mAb inhibits neutrophil homing in experimental colitis
Using the newly generated mCD97 mAb, we evaluated the role of CD97 in neutrophil migration. First, we investigated the homing of radioactively labeled neutrophils that had been incubated with mAb, to the colon in mice with experimental colitis. In this model, neutrophils, recruited from the peritoneum of donor mice and labeled with 99mTc-HMPAO, were incubated with cIg or 1B2 before adoptive transfer into the blood circulation of acceptor mice sensitized with DSS. As depicted in Fig. 2, homing of neutrophils treated with 1B2 to the colon but also to other sites was significantly diminished when compared with preincubation with cIg.
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Based on the delaying effect on neutrophil migration, we expected that application of mCD97 mAb would be detrimental for anti-bacterial immunity. This assumption was tested in the well established S. pneumoniae pneumonia model (43, 44, 45). We first assessed the effect of the mAb 1B2 and 1C5 on survival of mice after i.n. inoculation with 1 x 104 CFU S. pneumoniae. As shown in Fig. 3A, a dramatic difference was observed between mice treated with mCD97 mAb or cIg. Whereas all mice of the control group survived to day 20, 70% of animals from the 1B2- and the 1C5-treated group died within 2 wk.
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mCD97 mAb impair the recruitment of neutrophils to the lungs during pneumococcal pneumonia
Histological analysis revealed that at 20 h after infection with S. pneumoniae, lungs of all control mice displayed interstitial and peribronchial inflammation together with some degree of endothelialitis. Moreover, in 50% of these mice multiple foci of pneumonia were present in the lungs (Fig. 4A). In contrast, mice treated with mCD97 mAb presented less inflammation in the lungs and none of the animals had developed foci of pneumonia (Fig. 4, B and C). These findings were confirmed by immunostaining for granulocytes showing a prominent granulocytic infiltration in cIg-treated mice (Fig. 4D) but only few granulocytes in interstitial areas in mCD97 mAb-treated mice (Fig. 4, E and F). At 44 h after inoculation, all mice showed moderate inflammatory infiltrates essentially composed of monocytes and macrophages. There was no difference between control and treated mice (data not shown). The observation of similar degrees of infiltration in all three groups at 44 h may reflect a relative deficiency in neutrophil influx as the bacterial load was much higher in mCD97 mAb-treated mice at that time point.
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, IL-1) and neutrophil-attracting chemokines (KC, MIP-2) in the lungs did not significantly differ between the three groups. These data imply that alterations in the expression of humoral factors are unlikely to contribute to the diminished response in mCD97 mAb-treated mice during pneumococcal pneumonia.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In initial experiments, we adoptively transferred radioactively labeled neutrophils into mice with experimental colitis. Ex vivo incubation with mCD97 mAb significantly diminished homing, demonstrating that binding of mAb to CD97 on neutrophils causes a defect in the migration of these cells. Based on this observation, we expected that application of mCD97 mAb would diminish the resistance to bacterial infections. To test this idea, we used a murine model of S. pneumoniae-induced pneumonia. Application of mCD97 mAb caused an early and persistent defect in neutrophil migration to the lungs that was associated with the inability to control the growth of S. pneumoniae.
Together, these data reveal an important role of CD97 in the migration of neutrophils to sites of infection and injury. Neutrophils constitutively express substantial levels of CD97 (1, 6). After cellular stimulation, expression of CD97 on human neutrophils was found to increase 1.5- to 2-fold (20). Expression of CD97 on most types of myeloid cells and on activated lymphocytes (1, 23) indicates that the role of CD97 in leukocyte migration might not be restricted to neutrophils.
On current evidence, the observed effect of mCD97 mAb on neutrophil migration cannot be causally linked to one specific molecular interaction of CD97. Two cellular ligands of CD97 have been identified hitherto. EGF domains 1 and 2 mediate binding to the complement regulator CD55 (Refs.6 ,33 ,34 and this study). The more recently identified interaction with the GAG side chain chondroitin sulfate is mediated by EGF domain 4 in humans and its homologue in mice, EGF domain 3, respectively (Ref.38 and M. J. Kwakkenbos and J. Hamann, unpublished observation). For the in vivo experiments, mAb that recognize EGF domain 1 (1B2) and EGF domain 3 (1C5) were used. Despite different effects on the molecular interactions of CD97—1B2 blocks binding to CD55, whereas 1C5 is expected to interfere with chondroitin sulfate binding—application of both mAb had similar, impairing consequences for the course of pneumococcal pneumonia. This result might be explained in different ways. Possibly, mAb to the EGF-domain region, that do not block an interaction in cell-based adhesion assays, sterically hinder the same interaction in situ. Alternatively, both interactions could be engaged in the role of CD97 in neutrophil migration.
A possible interpretation for our data could be that CD97 facilitates the binding of chemokines to neutrophils. Chemokines are small, secreted proteins that are critically involved in the recruitment and activation of leukocytes (50). Divided into several families, different chemokines act on different types of leukocytes. For example, the most important neutrophil attractants are the glutamic acid-leucine-arginine-positive (ELR+) CXC chemokines KC and MIP-2 in the mouse and IL-8 in man (47). According to the classical view, chemokines are presented to leukocytes in two forms, either immobilized through binding to GAG at the endothelial extracellular matrix or as soluble molecules (51). This view has been challenged by studies showing that the formation of complexes with the GAG side chains of proteoglycans is generally needed for the in vivo activity of certain chemokines. Previously, it was found that IL-8 must bind GAG to elicit directed neutrophil migration (52). More recently, Li et al. (53) could demonstrate that the release of complexes between the cell surface proteoglycan syndecan-1 and the chemokine KC from the epithelium in lung injury is regulated by the matrix metalloproteinase matrilysin (MMP-7). In syndecan-1- or matrilysin-deficient mice, KC was hardly detectable in alveolar fluid and neutrophil influx was confined. Syndecan-1 is a component of the extracellular matrix. These findings indicate that extracellular matrix fragments cooperate with chemokines to create gradients that direct cell movement. Supportive evidence for this concept comes from an investigation showing that important monocyte attractants are devoid of their in vivo activity when the GAG-binding site is mutated (54). The question arises, whether leukocytes bind chemokines complexed with proteoglycans exclusively through chemokine receptors or whether additional molecules are involved. Based on its ability to bind chondroitin sulfate, it seems possible that CD97 has a role in the fixation of soluble proteoglycan-chemokine complexes on the leukocyte surface. Such a mechanism would support trafficking along chemokine gradients under the shear conditions caused by blood flow. In addition, it would promote the efficient parallel engagement of numerous chemokine receptors on individual cells.
In conclusion, this study for the first time provides in vivo evidence for a role for CD97 in cell migration. Whether the function of CD97 indeed relates to the binding of proteoglycan-chemokine complexes remains to be demonstrated in further investigations. In addition, it needs to be shown whether other EGF-TM7 receptors have a comparable role in leukocyte migration. Suggestively, EMR2, a molecular twin of hCD97 (not existing in mice) with a nearly identical EGF domain region (12), also binds chondroitin sulfate (38).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jörg Hamann, Laboratory for Experimental Immunology, G1-106, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail address: j.hamann{at}amc.uva.nl
3 Abbreviations used in this paper: EGF, epidermal growth factor; TM7, seven transmembrane; EMR, EGF module-containing mucin-like receptor; hCD97, human CD97; mCD97, mouse CD97; GAG, glycosaminoglycan; i.n., intranasal; DSS, dextran sulphate sodium; HMPAO, hexamethylpropylene amine oxime; MIP-2, macrophage-inflammatory protein-2; SPECT, single photon emission computed tomography.
Received for publication August 8, 2003. Accepted for publication October 21, 2003.
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  M. van Pel, H. Hagoort, J. Hamann, and W. E. Fibbe CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells Haematologica, August 1, 2008; 93(8): 1137 - 1144. [Abstract] [Full Text] [PDF]
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) or 1B2 (
). Medians are indicated with horizontal lines. Data are pooled from two separate experiments. n = 3–4/group/experiment. **, p < 0.05.
) was assessed 1–2 times daily for 20 days. The p value indicates the difference between the mCD97 mAb-treated groups and the control group, n = 10/group. B, Bacterial outgrowth in lungs. CFU S. pneumoniae in lungs of mice treated with cIg, 1B2, or 1D2 20 and 44 h after inoculation. Medians are indicated with horizontal lines, n = 8/group/time point. *, p < 0.05; ***, p < 0.0005.