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CUTTING EDGE |
*
Department of Microbiology, Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294; and
JCR Biopharmaceuticals, San Diego, CA 92121
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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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Native CT binds to monosialoganglioside (GM1) on epithelial cells and requires endocytosis followed by transport across the epithelial cell to reach the basolateral membrane where it induces water and chloride secretion for the characteristic cholera-type diarrhea (8, 9). These GM1 gangliosides are also abundantly expressed in the CNS. In this regard, the cholera toxin B subunit (CT-B) moiety of CT binds to GM1 gangliosides in membrane raft microdomains (10) on neuronal tissues (11) and is used as a standard technique for neuronal tracing of axonal pathways (12, 13, 14). Some studies have used CT-HRP to trace neuronal pathways within the olfactory system (15, 16).
In mammals, the sense of smell is performed by the main olfactory nerves and epithelium (ON/E), which is directly connected with the olfactory bulbs (OBs) as the first neural integrative center and with the olfactory cortex as the second integrative center (17, 18). The neuronal connections between ON/E and the OBs are used by pathogens for entry into the CNS. Although well established for viruses such as vesicular stomatitis virus (VSV) (19), no studies are available for the entry of bacteria or bacterial-derived toxins, such as CT, into olfactory CNS regions.
In this study, we tested whether CT and CT-B could bind to GM1 gangliosides expressed by the ON/E and, if so, whether they could track through retrograde transport to the OB and CNS. We also tested whether use of CT as intranasal adjuvant could potentially redirect vaccine proteins into the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free C57BL/6 mice were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were maintained under pathogen-free conditions in the University of Alabama at Birmingham Immunobiology Vaccine Center animal facility. The mice were 8–12 wk of age when used in all experiments.
Radioiodination of proteins
Recombinant CT-B (JCR Biopharmaceuticals, San Diego, CA), CT (List Biological Laboratories, Campbell, CA), tetanus toxoid (TT; Pasteur Merieux Connaught, Swiftwater, PA), and OVA (Sigma-Aldrich, St. Louis, MO) were radiolabeled with iodine-125 (125I). The radioiodination was performed with iodobeads (Pierce, Rockford, IL) for 10–12 min at room temperature as described previously (20). Free, unincorporated 125I was removed using a 10DG Bio-Rad desalting column (Bio-Rad, Hercules, CA) and by dialysis using a Slide Dialyzer (Pierce). The specific activities of the radiolabeled proteins were typically 1) for CT-B, 100 cpm/ng; 2) for CT, 109 cpm/ng; 3) for TT, 24.5 cpm/ng; and 4) for OVA, 132 cpm/ng.
To assess the ability of 125I-CT-B to bind GM1 ganglioside, 96-well microtiter plates (Microtest III; Becton Dickinson, Oxnard, CA) were coated with 3 µg/ml of GM1 (Sigma), washed, and blocked with PBS containing 1% BSA and 0.05% Tween 20. Increasing amounts of 125I-CT-B were added and incubated for 1 h at room temperature. A competitive inhibition-binding assay was used to determine the amount of 125I-CT-B and 125I-CT. The radiolabeled proteins were precipitated with 20% TCA (Fisher Scientific, Fairlawn, NJ) and the TCA-precipitable fractions (90–97%) were used for intranasal administration. The TCA-precipitable CT-B and CT fractions retained 60–70% and 90% of their GM1-binding properties after iodination, respectively. A bicinchoninic acid protein assay (Pierce) was used to determine the concentrations of radiolabeled proteins. To assess the ability of 125I-CT-B, 125I-CT, 125I-TT, or 125I-OVA to target the CNS following intranasal application, a total of 1.0–1.2 x 106 cpm were administered in PBS (10–12 µl volume, i.e., 5–6 µl/nare to naive mice).
Trafficking of radiolabeled proteins
125I-labeled CT-B, -CT, -OVA, or -TT was given intranasally to detect their presence in both lymphoid and CNS tissues. At 15 min (only CT-B), 1, 1.5, 2, 6, 12, and 24 h, and then daily for 6 days, the 125I levels present in various lymphoid and CNS tissues were determined. For lymphoid tissues, the nasopharyngeal-associated lymphoreticular tissue (NALT), cervical lymph nodes (CLN), spleen, and blood were assessed. The isolation of NALT and associated lymph nodes was performed as previously described (21). For isolation of CNS tissues, we examined ON/E, the OB, and the remainder of the brain. These tissues were isolated by separating the skull in a sagittal plane along the parietal and frontal bone plates. This was followed by the removal of both the frontal and nasal bone plates to gain access to the brain, the ON/E, and the OBs. The OBs, which constitute the frontal part of the brain, could be readily obtained after removal of the brain. The OBs exhibit nerves that exit the cranial cavity through the cribriform plate. The exiting nerves could be easily excised with the nasal epithelium (nasal turbinate) after removal of the nasal plates. The levels of cpm present in each tissue were determined by use of a scintillation counter.
Biotinylation of CT and CT-B and immunohistochemistry
Both CT-B and CT were biotinylated following dialysis in 0.1 M sodium bicarbonate buffer (pH 8.3) using a Slide Dialyzer (10-kDa m.w. cutoff). The dialyzed proteins were mixed with water-soluble biotin (biotin-X-NHS; Calbiochem, La Jolla, CA) for 2 h at room temperature. Free biotin was removed by gel filtration over a G25 Sephadex column. The OB and brain tissues were fixed in acetic acid-alcohol, and 4 µm paraffin sections were placed on poly-L-lysine-coated slides as routinely performed in our laboratory (22). To assess accumulation of CT-B-biotin in neuronal tissue following nasal application, an HRP staining procedure was used. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 30 min at room temperature. The slides were rinsed and incubated with streptavidin-HRP or avidin-biotin-complex (ABC) Vectastain (Vector Laboratories, Burlingame, CA) in a humidified chamber for 30 min at room temperature. The slides were rinsed and the color reaction was developed for 5–10 min using diaminobenzidine as substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The slides were counterstained with methyl green.
Statistics
The data are expressed as the mean ± one SE and the results compared by the unpaired Mann-Whitney U test. The results were analyzed using the Statview II statistical Program (Abacus Concepts, Berkeley, CA) adapted for MacIntosh computers.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the initial study, 125I-labeled CT-B was
given intranasally and both lymphoid tissues (NALT, CLN, MLN, and
spleen) as well as neuronal tissues (ON/E, OB, and brain) were analyzed
for the presence of 125I-CT-B. The CT-B was
detected in the peripheral blood within 15 min and represented 
50%
of the total counts administered intranasally (Fig. 1
A). The kinetics of
125I-CT-B clearance from blood and spleen were
similar and reached background levels by 48 h (Fig. 1A). Significant 125I-CT-B
accumulation initially occurred in the NALT and a small percentage
(<0.02%) remained associated with this tissue for 6 days (Fig. 1A). The 125I-CT-B associated with CLN
peaked at 1–2 h after intranasal application and was different from
the profile seen in blood, spleen, or NALT. The kinetics of
125I-CT-B in NALT with a slower decline in CLN
likely reflects a normal initial Ag-uptake pathway by NALT with
subsequent drainage into the CLN.
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B). The 125I-CT-B present in the OB
peaked at 15 min and remained relatively constant over time.
Furthermore, the ON/E and OBs were the only tissues that showed this
profile of 125I-CT-B accumulation. The levels of
125I-CT-B in the brain displayed a pattern
consistent with blood-associated CT-B, and
125I-CT-B levels were below 50 CT-B-specific cpm
by 24 h (Fig. 1B). The differences in CT-B association
with the neuronal tissues became even more apparent when the weight of
these organs and the total cpm associated with them were compared. The
CT-B levels in the OB were 34 times higher and the ON/E were 620
times higher than the radioactivity observed in the brain on a per
milligram basis (Fig. 1C). This indicates that specific
accumulation of CT-B in this organ presumably took place through GM1
binding, because no accumulation was observed after intranasal
application of either TT (see Fig. 4 below) or OVA alone (data not
shown).
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The distribution of 125I-labeled holotoxin
following intranasal application followed similar but slightly
different kinetics when compared with 125I-CT-B
(Fig. 2). First, the
125I-CT peaked later in blood, but had
significantly declined by 12–24 h after intranasal application.
However, most tissues including CLN, blood, spleen, and brain displayed
a delayed peak of 125I-CT that persisted longer
when compared with 125I-CT-B uptake. An exception
was NALT, which showed very similar kinetics of clearance as
125I-CT-B (Fig. 2A). Interestingly,
the ON/E bound higher levels of CT than CT-B and represented 1.7
µg of 125I-CT at 1.5 h. The levels of
125I-CT declined quickly within the first 24
h, and then fell more slowly over the following 6 days (Fig. 2B), a pattern also observed with
125I-CT-B. The binding of CT and CT-B in the OB
followed similar kinetics, and the levels of
125I-CT and 125I-CT-B
remained between 1.0 and 2.0 ng in the OB over the 6-day period tested
(Fig. 2B). Finally, this profile was noted with both
125I-CT-B and 125I-CT,
indicating that neuronal binding is a characteristic of CT.
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Immunohistochemical analysis of the CLN, OB, and brain was
performed following intranasal delivery of biotinylated CT-B. Maximum
accumulation of CT-B in the CLN had occurred by 1–2 h, and this was
consistent with the 125I-CT-B tracing experiments
(Fig. 1A). In contrast, CT-B tended to localize in the
olfactory nerves and the glomerular region over time (Fig. 3). This staining pattern clearly
suggested that retrograde axonal transport of CT-B occurred following
its intranasal application to mice. To test whether binding of
125I-CT-B to the OBs was GM1 specific,
125I-CT-B was preincubated with GM1 before
intranasal instillation. Preincubation of
125I-CT-B with a 13-fold molar excess of GM1
reduced (p 
0.0178) uptake into NALT and
olfactory regions by 94% at 48 h (data not shown).
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To determine whether CT, when used as mucosal adjuvant, redirects
protein vaccines into neuronal tissues, nonlabeled holotoxin was
coadministered with 125I-TT intranasally. The
125I-TT distribution in various tissues was
analyzed and was compared with 125I-TT given
alone. A delay in clearance of 125I-TT was
observed in both lymphoid and CNS tissues (Fig. 4). Elevated levels of
125I-TT were observed at 24 and 48 h and
this progressively decreased over the course of 6 days (Fig. 4).
Interestingly, significant accumulations of
125I-TT were observed in the ON/E between 12 and
48 h when 125I-TT was given with CT, when
compared with 125I-TT given alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have shown that both CT and CT-B accumulate in the ON/E and OB of mice when given by the intranasal route. Unlike CT or CT-B, no accumulation was observed with OVA or TT administered alone. This accumulation of CT-B and CT in the OB, which predominantly occurs in the olfactory nerve and glomerular layer of this tissue, could be inhibited by preincubating the molecule with its ligand GM1 before intranasal application. This data is consistent with a GM1-mediated retrograde axonal transport from the neurons of the olfactory epithelium to the OB tissue. No accumulation of CT-B was observed following intranasal application in CNS tissues other than the olfactory regions. Unlike CT-B, the accumulation of CT displayed a precipitous decline between 12 and 24 h in the ON/E and OB, which coincided with a decrease in blood-associated CT. However, the CT remained associated with the OB beyond 24 h at similar levels as seen with CT-B. Two possibilities could account for the rapid decline in 125I-CT in the OB at 24 h. First, it is possible that the 125I-CT was blood derived and thus exhibited a similar decline in radioactivity. A second more likely possibility was that CT was associated with inflammation and possibly apoptosis of neurons. The OB tissues displayed inflammatory responses of the meninges following a high intranasal dose of CT (30 µg; data not shown). The potential of enterotoxins to induce CNS damage was also supported by observations recently reported in Abstract form (26). In that study, intranasally applied LT of Escherichia coli induced inflammatory responses in meninges, the olfactory nerve, and glomerular layers of the OBs (26). In our study, CT-B accumulated in these same tissues following intranasal application. Thus, intranasal delivery of LT or CT derivatives targets the ON/E and the OB following intranasal application and raises the important possibility for neuronal damage with the intranasal use of these proteins in humans. In addition, these proteins may provide a means to induce protective immunity to meningitis-causing pathogens in the CNS following intranasal application of CT derivatives and vaccine Ags. Furthermore, microencapsulated therapeutic agents may be targeted to the brain by passing the blood-brain barrier after intranasal application of, e.g., CT-B-coated microspheres.
It is also important to emphasize that CT redirected trafficking of 125I-TT when given by the intranasal route. Although a more prolonged association of 125I-TT was observed with most tissues tested, the increase in 125I-TT was most striking and highly significant in the ON/E. We hypothesize that the use of CT as an adjuvant contributes to neuronal damage and inflammation in the ON/E, which then leads to an accumulation of coadministered protein Ag. This could contribute to adjuvanticity induced by CT and enterotoxins as well as by nontoxic CT and LT derivatives. After damage to the murine olfactory system, nerve growth factor (NGF) is produced by OB neurons and is retrograde transported to the olfactory cells through the olfactory nerves (27, 28). Thus, after damage of the ON/E, NGF accumulates in these sites and promotes survival and/or regeneration of neuronal cells. In an environment containing high levels of NGF and Ag as shown here, an infiltration of inflammatory cells and T lymphocytes would be expected to occur. In this regard, CD4+ Th0 and Th2 T cell clones have been shown to express NGF and its receptor after stimulation with Ag, whereas Th1 clones do not (29). Thus, activated Th0 or Th2 CD4+ T cells may be stimulated by NGF in an autocrine and/or paracrine fashion to regulate the immune response and induce potent Th2-type responses, as are seen when CT is used as mucosal adjuvant.
In summary, both radiolabeled CT-B and CT accumulate in the ON/E and to a lesser extent in the OB following intranasal application. In addition, when CT was used as a mucosal adjuvant, it redirected 125I-TT to the olfactory nerves and delayed systemic uptake and clearance of this vaccine protein. Furthermore, our results indicate a direct binding of CT-B to GM1-expressing olfactory neurons in the nasal olfactory epithelium with subsequent retrograde transport into the OB of the CNS. This conclusion is based upon the ability of GM1 to inhibit accumulation of CT-B in the OB as well as its accumulation in the outer layers of the OB, i.e., the olfactory nerve and glomerular layer.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry R. McGhee, Department of Microbiology, University of Alabama, Bevill Biomedical Research Building Room 761, 845 19th Street South, Birmingham, AL 35294-2170.
3 Abbreviations used in this paper: CT, cholera toxin; CLN, cervical lymph node(s); CT-B, cholera toxin B subunit; GM1, monosialoganglioside; NALT, nasopharyngeal associated lymphoreticular tissue; OB, olfactory bulb; TT, tetanus toxoid; ON/E, olfactory nerves and epithelium; LT, labile toxin; NGF, nerve growth factor.
Received for publication July 18, 2000. Accepted for publication August 24, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. M. I. Mowat, A. M. Donachie, S. Jagewall, K. Schon, B. Lowenadler, K. Dalsgaard, P. Kaastrup, and N. Lycke CTA1-DD-Immune Stimulating Complexes: a Novel, Rationally Designed Combined Mucosal Vaccine Adjuvant Effective with Nanogram Doses of Antigen J. Immunol., September 15, 2001; 167(6): 3398 - 3405. [Abstract] [Full Text] [PDF]
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S. Donnelly, C. E. Loscher, M. A. Lynch, and K. H. G. Mills Whole-Cell but Not Acellular Pertussis Vaccines Induce Convulsive Activity in Mice: Evidence of a Role for Toxin-Induced Interleukin-1{beta} in a New Murine Model for Analysis of Neuronal Side Effects of Vaccination Infect. Immun., July 1, 2001; 69(7): 4217 - 4223. [Abstract] [Full Text] [PDF]
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) or
without (
) CT (1 µg) as mucosal adjuvant. A, Uptake
of TT into NALT, CLN, blood, and spleen over a 6-day period.
B, Distribution of 125I-TT in ON/E, OB, and
CNS. The results are taken from three mice per group and are
representative of three separate experiments.
