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Department of Surgery, University of Tennessee, Memphis, TN 38163
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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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70% of the body’s total Ig production (1). B and T
cells sensitized to intraluminal Ags within the Peyer’s patches (PP)
of the GALT and the draining lymph nodes of the bronchus-associated
lymphoid tissue (2, 3, 4) ultimately home to the lamina
propria (LP) of both intestinal and extraintestinal sites, where they
carry out their mature immune function. IgA produced by LP plasma cells
is transported by a secretory component (SC)-mediated process through
the mucosal epithelial cells into the mucocilliary blanket coating
moist mucosal surfaces, such as the respiratory and gastrointestinal
(GI) tracts, where it affords protection by neutralizing or otherwise
preventing the attachment of viruses, bacteria, and toxins to the
mucosal epithelium, thus allowing luminal clearance of these
potentially pathogenic agents (5, 6, 7, 8, 9). IgA has been shown
to be responsible for the protection of the intact murine nose against
influenza infection in immune mice (7, 10). Recently, this
finding has been challenged by studies in IgA knockout (KO) mice
suggesting that other Igs are as effective as IgA in the protection of
the murine nose and that IgA may not be necessary for mucosal
protection (11). The KO mouse, while an interesting model, is genetically abnormal. We offer in this report a model for the suppression of mucosal anti-influenza immunity in normal, immune mice. Intravenous feeding with a chemically defined solution (i.v. total parenteral nutrition, TPN) results in a down-regulation of the murine common mucosal immune system with significant reductions in the numbers of both B and T cells present within the PP, LP, and intraepithelial (IE) region of the GI tract (12, 13) and lowered secretory IgA (SIgA) levels throughout the mucosal immune system (12, 13, 14). Impaired mucosal immunity is observed in both the upper (URT) and lower (LRT) respiratory tracts of i.v. TPN-fed mice (12, 15, 16), and alterations in GI IL-4 and IL-10 levels have been reported (17). The impairment in immunity is reversed either by 3 days of oral alimentation (14) or by injection of i.v. TPN-fed mice with the neuropeptide bombesin (18, 19), which mimics human gastrin-releasing peptide in stimulating the release of all other GI neuropeptides (20). We propose i.v. TPN feeding as a model for the suppression of mucosal immunity in genetically normal mice.
The purpose of this study, then, is to determine in a model of impaired mucosal immunity in genetically normal immune mice whether serum IgG can substitute for SIgA in mucosal immunity against influenza virus in the murine URT.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old male ICR mice (Harlan Sprague Dawley, Indianapolis, IN) were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited conventional facility under controlled conditions of temperature and humidity with a 12-h light, 12-h dark cycle. Before the experiment, mice were fed water and commercial chow ad libitum. During the experiments, the mice were housed in metal, wire-grid bottom metabolism cages to eliminate coprophagia and the ingestion of bedding. All protocols were approved by the institutional animal care and use committee.
Immunization and challenge
For immunization, 6- to 8-wk old male ICR mice were inoculated intranasally (i.n.) while awake with 20 µl of PBS (pH7.4) containing 100 mouse 50% infectious doses (MID50) of A/PR8 (H1N1) influenza virus. For challenge, immune mice were inoculated in the same manner. Administration of virus to awake mice leads to infection of the URT only and does not produce fatal pneumonitis.
Surgical procedures
Mice randomized into i.v. TPN and chow groups received internal jugular catheters under ketamine/acepromazine anesthesia (100 and 10 mg/kg, respectively, i.p.). As previously described (12), through a right internal jugular approach a silicone catheter (0.012-in. inside diameter by 0.025-in. outside diameter; Baxter, Chicago, IL) was placed into the vena cava. The catheter was tunneled s.c. and exited the tail at its midpoint. The mice were placed into metal metabolism cages, partially immobilized by tail restraint to protect the catheter during infusion, and immediately connected to an infusion apparatus (720 infusion pump, Instech, Plymouth Meeting, PA), and physiologic saline solution was infused at an initial rate of 4 ml/day. This technique is an acceptable method of nutritional support that does not induce physical or biochemical stress (21).
Intragastric (i.g.) TPN group
For intragastic feeding, anesthetized mice received gastrostomy tubes as previously described (12). Through a vertical midline incision, the stomach was delivered into the wound. A 7-0 silk purse-string suture was placed on the posterior wall of the stomach, followed by a gastrostomy within the center of the purse-string suture using a 25-gauge needle. The silicone catheter was inserted into the gastrostomy. Omentum was mobilized and free-tied around the insertion of the gastrostomy tube using 7-0 silk suture. The proximal end of the catheter was tunneled s.c. over the spine and exited the tail at its midpoint. As with the other two groups, catheters were immediately connected to an infusion pump delivering physiologic saline at 4 ml/day.
Mice receiving venous catheters underwent sham laparotomies, and those receiving gastrostomy tubes underwent sham neck incisions.
All animals were allowed ad libitum access to chow for 2 days, then were placed on their respective treatment protocols for an additional 5 days unless noted otherwise. The chow-fed group received 4 ml of 0.9% saline i.v. daily in addition to mouse chow and water ad libitum. The i.v.-fed and i.g.-fed TPN groups received a standard TPN solution (4.1% amino acids, 34.3% glucose, electrolytes, and multivitamins with a nonprotein calorie to N ratio of 740 kJ/g N), which was initially given at the rate of 4 ml/day, but was increased over the next 24 h to 10 ml/day to meet energy and protein requirements (1619 kJ/kg/day of nonprotein calories and 14 g protein/kg/day as previously published (12)).
Sample collection
Nasal washes (NWs) were collected as previously described (7, 10). Briefly, a midline incision was made over the ventral aspect of the trachea slightly anterior to the thoracic inlet. The trachea was clamped off at the thoracic inlet, and 400 µl (Ab assays) or 500 µl (viral assays) of PBS was slowly injected into the tracheal lumen cephalad to the obstruction by using a 25-gauge B bevel needle attached to a tuberculin syringe. Care was taken to avoid blood contamination of the nasal lavages. NWs for viral assay were placed on ice upon collection and immediately assayed for influenza virus. NWs for ELISA analysis were placed upon ice immediately upon collection and stored at 4°C until assay. Blood was collected via cardiac puncture, and the serum was retained for Ab determination.
Virus
A/PR8-Mt. Sinai (H1N1) influenza virus was the gift of Parker A. Small, Jr. (University of Florida, Gainesville, FL). To generate a pool, the virus was grown in fertile specific pathogen-free eggs, pooled, filtered through a 0.45-µm pore size filter, aliquoted, and stored at -70°C. The 50% lethal dose for mice was established by the i.n. inoculation of 50 µl of 10-fold serial dilutions of the virus pool into anesthetized mice. The log10 50% mouse lethal dose of the virus pool was 5.
ELISA
A modification of the ELISA described by Renegar and Small (10) was used. A/PR8 (H1N1) influenza vaccine (the gift of Dr. Frank Brandon, Parke Davis, Rochester, MI) in PBS (pH 8.1) at a 1/25 dilution was dried onto 96-well flat-bottom enzyme immunoassay microtiter trays (Linbro/Titer-Tek E.I.A. II microtitration plate, Flow Laboratories, Rockville, MD). Trays were blocked for 1 h with PBS-Tween containing 1% BSA (BSA for ELISA diluent applications from Sigma, St. Louis, MO) and 1% normal rabbit serum. Ab-containing samples were incubated in wells overnight, the plates were washed, and the secondary and tertiary Ab were added and incubated for 1.5 h with appropriate washings between Ab additions. Affinity-purified goat anti-mouse IgA or IgG (Sigma) was the secondary Ab, and the final Ab was rabbit anti-goat IgG conjugated to alkaline phosphatase (Sigma ). All Abs were diluted in PBS-Tween containing 1% BSA and 1% normal rabbit serum. PBS-Tween was used for all plate washes. Only specific, biologically active Ab capable of binding influenza virus is detected in this assay.
Viral assay
A modification of the viral assay described by Bender et al. (22) was used. Viral samples were serially diluted (10-fold) in DMEM supplemented with 2.5 µg/ml amphotericin B (Sigma), 50 µg/ml gentamicin, and 10% FCS. Triplicate 100-µl samples of each dilution were placed into 96-well round-bottom tissue culture plates. To each well was added 100 µl of a 2 x 105 cells/ml suspension of MDCK cells in supplemented DMEM-10% FCS. The plates were incubated 24 h at 34°C in 5% CO2. The culture fluid was removed and replaced with DMEM (150 µl/well) containing 2.5 µg/ml amphotericin B, 50 µg/ml gentamicin, and 2 µg/ml trypsin (DMEM-trypsin). The plates were incubated 4 days at 34°C in 5% CO2. Assay for viral growth was by hemagglutination. To each well was added 50 µl of a 0.5% suspension of chicken RBC. Hemagglutination was read after 1–2 h in the cold. Viral titers were calculated by the method of Reid and Muench (23).
Spleen and nasal passage (NP) lymphocyte preparation
Mice were anesthetized with ketamine-acepromazine to effect, exsanguinated via the retro-orbital plexus, then decapitated. The facial skin, cheek muscles, and palate were removed, and the NP lymphocytes obtained from the remaining nasal tissues (nasal turbinates, septa, and lateral walls) by a modification of the technique described by Asanuma et al. (24) and Hiroi et al. (25). The NP tissues from each individual mouse were cut into small pieces in culture dishes containing 2 ml of IMEM supplemented with 4 mg/ml collagenase (Sigma), transferred to individual 15-ml centrifuge tubes, and incubated for 30 min at 37°C. After incubation, dissociated cells were filtered and washed twice in IMEM, then resuspended in 2 ml of IMEM containing 75% Percoll (Pharmacia, Uppsala, Sweden). Four milliliters of a 40% Percoll solution were layered on top of the 75% layer, and the interface was collected after centrifugation at 600 x g for 20 min at 25°C. For preparation of splenic lymphocytes, spleens were pressed through a 40-gauge wire mesh, the resultant cells were suspended in 5 ml of IMEM-10% FCS and centrifuged 5 min at 400 x g, and the pellet was resuspended in 10 ml of IMEM-10% FCS. Viable lymphocytes from each source for each individual mouse, as determined by trypan blue exclusion, were suspended in IMEM-10% FCS and assayed for Ab-forming cells (AFCs) by the enzyme-linked immunospot (ELISPOT) assay described below.
ELISPOT procedure
The ELISPOT assay was performed as described by Sedgewick and
Holt (26). Briefly, 96-well nitrocellulose-bottom plates
(Millipore, Bedford, MA) were coated overnight with affinity-purified
goat anti-mouse 
and 
Ab (Southern Biotechnology Associates,
Birmingham, AL), washed in PBS, and blocked with 1% BSA in PBS.
Lymphocytes (4 x 104 to 2 x
105/well) suspended in IMEM-10% FCS were added
to duplicate wells and incubated 4 h at 37oC
in 5% CO2. The plates were washed with PBS-Tween
and incubated overnight at 4°C with 50 µl of goat anti-mouse
IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA conjugated with alkaline
phosphatase (Southern Biotechnology Associates). Plates were washed
with PBS, and spots representing single Ab-secreting cells were
developed with 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in
diethanolamine buffer. Optimal spot development generally required
1 h at room temperature, after which the plates were washed with
tap water and allowed to dry overnight. Blue spots representing Ab
secretion by individual cells were counted with the aid of a dissecting
microscope.
Experimental design
Expt. 1: impairment of nasal mucosal immunity by TPN. In a series of experiments, 123 mice were randomized to one of four groups: chow-fed immune (n = 38), i.g. TPN-fed immune (n = 10), i.v. TPN-fed immune (n = 51), or nonimmune (n = 24). Mice were instrumented as described above and, after 5 days on their respective feeding protocols, were challenged awake i.n. with 100 MID50 influenza virus. After 42 h, mice were sacrificed by exsanguination under ketamine/xylazine anesthesia, and viral shedding in their nasal secretions was determined. These data represent the pooled results from 17 experiments.
Expt. 2: levels of serum IgG in impaired mice. Twenty-two mice were immunized i.n. with A/PR8 influenza virus. Three weeks later, the mice were randomized to chow-fed, i.g. TPN-fed, or i.v. TPN-fed groups and instrumented as described above. At the time of surgery, serum samples were obtained for determination of influenza-specific serum IgG levels. Mice were allowed to recover for 2 days, then underwent the described feeding protocols. After 5 days on a protocol, they were challenged i.n. awake with 100 MID50 A/PR8 influenza virus, then sacrificed 42 h later, and viral shedding in the nasal secretions was determined. Viral titers were calculated by the method of Reid and Muench (23) and were correlated graphically with preinstrumentation serum influenza-specific IgG levels. For the sake of this correlation, serum IgG-specific Ab levels were taken to be the OD405 nm of a 1/100 dilution of the serum sample.
Expt. 3: effect of TPN on serum IgG levels. Eleven mice immunized 3 wk previously were instrumented as described and fed either i.v. saline with ad libitum chow access or i.v. TPN. Serum samples were taken at the time of initial surgery. The mice were allowed to recover from surgery for 2 days, then underwent 5 days of their respective feeding protocols and were challenged i.n. while awake with 100 MID50 A/PR8 influenza virus. After 42 h, the mice were sacrificed under ketamine/xylazine anesthesia and exsanguinated by cardiac puncture, and nasal lavages were performed for viral assay. Serum samples from the time of sacrifice were saved for Ab determination. Serum IgG anti-influenza titer was taken as the serum dilution providing an ELISA OD405 nm of 0.200.
Expt. 4: nasotracheal (NT) influenza-specific SIgA levels. Sixteen immune mice were surgically instrumented as described. Following the 2-day recovery period, they were randomized to one of two groups and fed either chow ad libitum (n = 7) or TPN solution i.v. (n = 8). The chow group also had i.v. lines for continuous saline administration. After 5 days of their respective protocols, mice were sacrificed by exsanguination under ketamine/xylazine anesthesia. NT lavages were performed, and the resulting washes were assayed for influenza-specific IgA by ELISA. NW-specific IgA results are expressed as nanograms of specific IgA per 100 µg of total nasal lavage protein. Our previously described polymeric IgA (pIgA) anti-influenza mAb pool served as the IgA standard (27). The protein concentration of the nasal secretions was determined using a micro protein assay (Bio-Rad, Hercules, CA), and protein and specific IgA levels were normalized to 100 µg of total protein/ml of lavage fluid.
Expt. 5: spleen and nasal mucosal ELISPOT AFCs. Thirteen nonimmune male ICR mice were surgically instrumented as described above and fed chow and i.v. saline for 2 days. They were then randomized to either saline i.v. with chow ad libitum (n = 5) or i.v. TPN (n = 8) groups and were inoculated i.n. while awake with 100 MID50 influenza virus. Following 13 days of feeding on their respective protocols, the mice were sacrificed, NP and spleen cells were obtained as described, and ELISPOT assays for AFCs were performed. These animals were given influenza virus to stimulate proliferation of lymphoid cells within the NPs. Our data are from individual animals, not pooled cells, and we were unable to obtain an adequate number of cells from single animals without on-going immune stimulation. ELISPOT assays were for total AFCs of each Ab class, not for influenza-specific AFCs, because we wanted to determine the effects of TPN on the general AFC population. Day 13 was determined in a pilot study to be the best time point for the enumeration of Ig spot-forming cells by ELISPOT.
Statistics
All data are expressed as the mean ± SE unless otherwise indicated. Nonparametric (Mann-Whitney) and parametric (unpaired two-tailed Student’s t test, ANOVA, and Fisher’s protected least significant difference) analyses were conducted on a Macintosh Performa using StatView 4.2 (Brain Power, Calabasas, CA) software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of i.v. TPN feeding on URT mucosal immunity are well
established. In a series of 17 experiments (summarized in Fig. 1
), chow-fed (n = 38),
i.g. TPN-fed (n = 10), and i.v. TPN-fed
(n = 51) immune mice or chow-fed (n =
24) nonimmune mice were infected i.n. while awake with 100
MID50 influenza virus. As expected, chow-fed
immune mice were protected (2 of 38, or 5.3%, shed virus), while
nonimmune mice were not (22 of 24, or 91.6%, shed virus;
p < 0.0001 vs chow-fed immune). The i.v. TPN-fed
immune mice lost nasal mucosal immunity against influenza virus (30 of
51, or 58.8%, shed virus; p < 0.0001 vs chow-fed
immune), while immune mice fed the same diet directly into the GI tract
via a gastrostomy (i.g. TPN-fed) maintained their immunity against
influenza virus (0 of 10 shed virus; p < 0.4 vs
immune; p < 0.005 vs i.v. TPN-fed). These results show
that i.v. TPN feeding depresses nasal mucosal immunity against
influenza virus and that the loss of immunity is related to the route
of feeding, not the composition of the diet, because the same diet fed
via gastrostomy maintains immunity. The consistent loss of nasal
immunity in TPN-fed mice throughout the series of experiments
summarized herein emphasizes the validity of our model as a tool for
the investigation of impaired mucosal immunity in genetically
normal mice.
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Mbawuike et al. (11) suggested that serum
influenza-specific IgG is sufficient to protect the murine nose against
influenza infection. To determine whether this was so in our model, 22
immune mice undergoing surgical instrumentation were screened for serum
influenza-specific IgG. The catheterized mice were placed on a feeding
protocol for 5 days, then challenged i.n. awake with influenza virus.
We compared the presurgery anti-influenza IgG ELISA reading of a
1/100 serum dilution with protection against challenge (Fig. 2). All immune chow-fed
(n = 8) or i.g. TPN-fed (n = 7) mice
were protected against influenza infection, while four of seven (57%)
immune i.v. TPN-fed mice shed virus. The serum influenza-specific IgG
readings of the protected mice ranged from 0.255 to 0.832, while those
of the four i.v. TPN-fed immune mice shedding virus were 0.263, 0.567,
0.641, and 0.786. Thus, all mice studied had humoral immunity to
influenza virus at the onset of the feeding protocol, and there was no
relationship between serum influenza-specific IgG levels and nasal
mucosal protection.
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It is possible that the i.v. administration of TPN could alter
serum Ab titers; therefore, a second experiment to determine the effect
of i.v. TPN on serum influenza-specific IgG titers was conducted. Serum
was taken from 11 immune mice at the time of surgical catheterization.
The mice underwent either 5 days of i.v. TPN feeding (n
= 6) or 5 days of chow feeding with continuous saline infusion i.v.
(n = 5), were challenged i.n. awake with influenza
virus, and were sacrificed 42 h later. Additional blood samples
were taken at the time of sacrifice for the comparison of pre- and
post-treatment influenza-specific serum IgG titers, and nasal
secretions were analyzed for viral shedding. The results are shown in
Table I. The i.v. TPN feeding did not
lower serum influenza-specific IgG titers. The mean titer before TPN
administration was 2133 ± 592, while that after TPN
administration was 3250 ± 1109 (p for titer
change = 0.05). The observed increase in titer is probably a
manifestation of the normal on-going immune response. The mice used in
this study are convalescent mice initially infected i.n. with influenza
virus 3 wk before TPN feeding; therefore, it is normal for the serum
anti-influenza titers to be on the rise at this point. Serum
influenza-specific titers of chow-fed, i.v.-saline-treated animals
exhibited a similar pattern during the feeding protocol, with a mean
final titer of 4180 ± 1543 (p < 0.25 vs
TPN-fed final titer). All TPN-fed mice in this experiment shed virus in
their nasal secretions, while none of the chow-fed mice shed virus
(p < 0.01). There was no relationship between
the serum influenza-specific IgG titer and the level of virus
shedding.
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Because serum influenza-specific IgG levels did not correlate with
the loss of URT protection against influenza challenge, studies were
undertaken to determine whether the loss of URT antiviral mucosal
immunity could be due to lowered anti-influenza SIgA Ab. Immune
mice were surgically implanted with an i.v. catheter, allowed to
recover from surgery, randomized to chow-fed or i.v. TPN-fed groups,
placed on a feeding protocol for 5 days, then sacrificed via cardiac
exsanguination under anesthesia. Levels of influenza-specific SIgA Ab
in the undiluted nasal lavages were assessed via ELISA and expressed as
nanograms of influenza-specific IgA Ab per 100 µg of total protein
(Table II). Nasal secretions of the
chow-fed immune group (n = 7) had a mean
influenza-specific IgA Ab level of 2218 ± 794 ng/100 µg
protein, while those of i.v. TPN-fed immune mice (n =
8) had a mean value of 388 ± 128 ng/100 µg protein. Nasal
influenza-specific SIgA in the TPN-fed group was significantly lower
than that in the chow-fed group (p < 0.05).
These results suggest that lowered SIgA in the nasal secretions may
underlie the immune deficit in i.v. TPN-fed mice.
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ELISPOT AFC in the spleens and NPs of TPN-fed mice
To determine whether i.v. TPN affects the AFC of the NPs and
spleen, 13 nonimmune mice were cannulated as described and randomized
to either chow (n = 5) or i.v. TPN (n =
8) groups. To stimulate the NP cells to proliferate to a number
detectable in individual animals, 100 MID50
influenza virus was administered i.n. to each mouse. Thirteen days
later, mice were sacrificed, NP and spleen cells were isolated, and the
number (Fig. 3) and relative percentage
(Fig. 4) of AFCs in each animal were
determined by ELISPOT analysis.
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) in either the NPs
(Fig. 3A; TPN, 89 ± 22 AFC/106
cells; chow, 99 ± 31 AFC/106 cells;
p < 0.8) or spleen (Fig. 3B; TPN, 1612
± 388 AFC/106 cells; chow, 1457 ± 599
AFC/106 cells; p < 0.8) of
treated mice; however, IgM AFCs were increased marginally, although not
significantly, in both the NPs and spleens of TPN-fed mice, and IgA
AFCs were decreased. The total number of IgG-producing cells in the
spleen (Fig. 3B; TPN, 735 ± 163
AFC/106 cells; chow, 715 ± 278
AFC/106 cells; p < 0.95) was
unaffected by i.v. TPN feeding.
The relative percentages of IgM- and IgA-producing AFC underwent a
minor shift with TPN treatment (Fig. 4). In the NPs (Fig. 4A), the percentage of IgA-producing AFCs fell from 40% in
chow-fed to 32% in TPN-fed mice (p < 0.6),
while the percentage of IgM-producing AFCs rose from 17% in chow-fed
to 21% in TPN-fed mice (p < 0.7). Similar
changes were seen in the spleen (Fig. 4B), with the
percentage of IgA-producing AFCs falling from 14% in chow-fed to 6%
in TPN-fed mice (p = 0.11) and the percentage
of IgM-producing AFCs rising from 36% in chow-fed to 46% in TPN-fed
animals (p = 0.35). The relative percentage of
IgG-producing cells rose slightly in the NPs and fell slightly in the
spleen with TPN feeding; however, the changes were minimal.
Although the changes in cell number and relative percentages of AFCs of each class are not statistically significant, the changes in the number and percentage of IgA-producing cells, especially in the spleen, are suggestive of a possible effect of TPN. IgG-producing AFCs, especially those in the spleen, do not appear to be adversely affected by TPN feeding.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protection of the URT of normal BALB/c mice has been shown to be due to
IgA. In a series of experiments reported in 1991, Renegar and Small
(7, 10) demonstrated that influenza-specific pIgA mAb
administered i.v. to nonimmune mice is preferentially transported into
nasal secretions relative to influenza-specific IgG mAb, strongly
suggesting a role for SC in pIgA transport. Intravenously administered
influenza-specific pIgA mAb, but not i.v.-administered IgG mAb, was
able to protect the noses of these mice against influenza infection.
Furthermore, the nasal protection provided by the passively
administered pIgA could be eliminated by the i.n. administration of
anti-
, but not anti-
, antiserum. In
influenza-convalescent mice immune to infection with influenza virus,
nasal mucosal immunity could be eliminated by the i.n. instillation of
anti-, but not anti- or anti-µ, antiserum. The
authors concluded that mucosal protection in the intact noses of normal
BALB/c mice is due to SIgA Ab. Epstein et al. (28) did not
assess immunity in the noses of J chain KO mice, and the Mbawuike study
(11) nasal turbinate and NW passive transport data, while
suggesting that Igs other than IgA can mediate protection in the KO
mouse nose are somewhat equivocal, because no attempt was made to
control for the contamination of the samples by endogenous blood. In
passively immunized animals, the high Ab levels reached in the blood
(especially of monomeric Ig such as IgG) can contaminate lavages or
tissue homogenates and neutralize virus present in the sample, causing
false neutralization readings.
The IgA KO mouse provides an excellent model for study of the host defenses that become functional in the absence of IgA and is a good model for those cases in which humans express a selective IgA deficiency. As in those humans, the immune system in this mouse must compensate for the loss of a major component and apparently does so by the production of higher levels of IgG. However, the congenital removal of IgA may have additional, as yet unrecognized consequences for Ab production and transport within the mucosal immune system. Thus, the responses seen in these mice may not be those observed in the normal population, and care must be taken not to overinterpret KO data. Much evidence exists to support the importance of IgA for the mucosal protection of genetically normal individuals. In this report we present a model for the impairment of nasal mucosal immunity in genetically normal mice and show once again the importance of SIgA in the protection of the murine nose against influenza infection.
We have previously reported that feeding of mice parenterally rather
than enterally has deleterious effects on the common mucosal immune
system. GALT atrophy occurs with associated decreases in PP cells, LP
lymphocytes, and IE lymphocytes (IEL) (12); total SIgA
levels decrease in the GI (12) and respiratory
(14) tracts; and functional mucosal immunity is impaired
in both the URT (14, 15, 18) and LRT (16, 19). Anti-influenza mucosal immunity is maintained in the URT of
mice fed TPN solution i.g. (Figs. 1 and 2), demonstrating that the
chemically defined solution fed is able to provide adequate nutrition
for the support of mucosal immunity and that the route by which the
solution is fed is of primary importance. Furthermore, as we have shown
in studies reported previously, the adverse effects of i.v. TPN on the
mucosal immune system can be prevented or reversed by the injection of
the neuropeptide bombesin (18, 19), an amphibian analogue
of human gastrin-releasing peptide (20), so the effect
cannot be attributed to malnutrition. The neuropeptide bombesin
stimulates the release of the GI neuropeptides normally released by the
presence of food within the GI tract; presumably, chow and the i.g.
feeding of TPN solution have the same function.
In this report we show that influenza-specific SIgA in the NT lavages
of TPN-fed genetically normal immune mice (Table II) is 18% that in
chow-fed mice and that this decrease mirrors the impairment of
protection against i.n. influenza virus infection seen in TPN-fed mice
(Fig. 1). In contrast to the KO mouse findings, serum
influenza-specific IgG Ab does not compensate for the loss of
influenza-specific nasal SIgA in TPN-fed mice (Fig. 2). Intravenous TPN
feeding does not suppress serum influenza-specific IgG, but does
depress nasal mucosal immunity. In five of six TPN-fed mice (Table I),
the serum IgG anti-influenza titer continued to rise during i.v.
feeding, reaching a level comparable to that seen in catheterized
immune chow-fed mice; however, unlike the chow-fed immune mice, these
TPN-fed immune mice were not protected against influenza infection of
the nose.
We have shown by ELISPOT analysis that prolonged i.v. TPN feeding has
no adverse effect on the number of IgG-producing cells in the spleen
(TPN, 735 ± 163 cells/106 spleen cells;
chow, 715 ± 278 cells/106 spleen cells).
Both the number and the relative percentage of IgA APC in the spleens
of TPN-fed mice are depressed relative to those in chow-fed mice (Figs. 3 and 4), however. This depression, although not statistically
significant (number, p = 0.18; percentage,
p = 0.11), is highly suggestive and is consistent with
the reduction in systemic IgA production previously reported in TPN-fed
mice (12, 14), agreeing with the hypothesis that
IgG-mediated humoral immunity remains intact in TPN-fed mice, while
IgA-mediated immunity is impaired. The 8% drop in the relative
percentage of IgA AFCs in the NP of TPN-fed mice compared with chow-fed
mice also supports this hypothesis. The presence of a normal number of
IgG- and IgM-producing cells in the NP of TPN-fed mice further
demonstrates that IgG and IgM do not substitute for SIgA in the
genetically intact mouse, because these cells are presumably also
present and producing Ab in the LP of immunologically impaired mice,
but protection against URT influenza infection is lost.
The mechanism for the loss of IgA-mediated mucosal immunity in TPN-fed
mice has not yet been fully elucidated; however, modulation of both the
production and transport of mucosal Ab is likely to be involved. We
have previously shown a drop in GALT PP, IEL, and LP lymphocyte yields
in TPN-fed mice, with both B and T cells being affected
(8). The LP
CD4+/CD8+ T cell ratio
decreased in TPN-fed mice, primarily due to a drop in
CD4+ cells, although the PP and IEL ratios
remained unchanged. The B cell yield in all three GALT compartments of
TPN-fed mice was reduced to 60% that of chow-fed animals, as were
the levels of IgA within gut and serum. It is possible that the factors
leading to the atrophy of the GALT in TPN-fed mice could also exert an
influence on the IgA AFCs of the NPs.
We have shown previously (27) that the selective
transport of i.v. administered pIgA is lowered by 75% in TPN-fed
mice compared with that in chow-fed mice, suggesting that impaired
transport of IgA into secretions makes a major contribution to the
lowered influenza-specific SIgA levels and decreased nasal
anti-influenza mucosal immunity seen in TPN-fed animals.
Furthermore, we have shown that protection can be restored if the
defective transport system is saturated by i.v. administered pIgA
anti-influenza mAb (27), suggesting that lowered
production of IgA Ab in the LP, discussed above, also contributes to
the defect in nasal mucosal immunity, because IgA-mediated protection
can be restored if adequate amounts of Ab are available for
transport.
The impaired transport of pIgA (27) suggests that
expression of SC may be depressed in TPN-fed animals. This is not
unexpected, because SC expression is known to be under cytokine
control. SC expression in intestinal and uterine epithelial cells is
regulated by IFN- (30, 31, 32), the expression of membrane
SC is enhanced in a synergistic fashion by IFN- and IL-4
(33), and SC production, surface expression, and release
can be up-regulated in colonic cell lines by TNF- (34).
We have shown (17) that although the level of IFN-
remains unchanged by diet, the levels of IL-4 and IL-10 in the
intestinal tracts of TPN-fed mice decrease significantly, with this
decrease correlating to a decline in intestinal IgA levels. We
hypothesize that in TPN-fed animals IL-4:IFN- synergism is lacking,
so a lowered level of SC expression is to be expected. In enterally
nourished chow-fed mice, normal IL-4:IFN- interaction occurs,
allowing for normal SC expression. Further studies are required to
substantiate this hypothesis.
In conclusion, the data reported herein support the previous body of work demonstrating the importance of IgA in nasal anti-influenza mucosal immunity (7, 10) and reiterate the importance of vaccination strategies that trigger Ab production at mucosal surfaces. In addition to its obvious importance for the study of the effects of GI neuropeptides on mucosal immunity, our model has significant clinical implications. Nosocomial respiratory tract infections, such as bacterial or viral pneumonia, account for 100,000 deaths annually in the United States alone and are the second most common hospital-acquired infection (35, 36, 37). Clinically, severely injured patients fed via the GI tract have a significantly lower incidence of pneumonia than patients fed i.v. (38, 39, 40). It may be that the clinical use of pharmacologic agents such as the neuropeptide bombesin, which has been shown to restore TPN-impaired mucosal immunity in mice (13, 18, 19), will lead to a decrease in the morbidity and mortality due to nosocomial pneumonia in severely injured patients. In addition, the respiratory mucosal surfaces of patients undergoing prolonged TPN may be potential candidates for protection by the passive administration of protective pIgA Ab.
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Footnotes |
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2 K.B.R. and C.D.J. contributed equally to this publication.
3 Address correspondence and reprint requests to Dr. Kenneth Kudsk, Department of Surgery, University of Tennessee, 956 Court Avenue, Room E228, Memphis, TN 38163.
4 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; AFC, Ab-forming cell; ELISPOT, enzyme-linked immunospot; GI, gastrointestinal; IE, intraepithelial; i.g., intragastric; i.n., intranasal(ly); IEL, IE lymphocytes; KO, knockout; LP, lamina propria; LRT, lower respiratory tract; MID50, mouse 50% infectious dose; NP, nasal passage; NW, nasal wash; pIgA, polymeric IgA; PP, Peyer’s patches; SC, secretory component; SIgA, secretory IgA; TPN, total parenteral nutrition; URT, upper respiratory tract; NT, nasotracheal.
Received for publication February 16, 2000. Accepted for publication October 16, 2000.
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), i.g. TPN (n = 5; 
), or i.v. TPN
(n = 7; 57 ). Serum samples were collected at the
time of surgery and assayed for influenza-specific IgG via ELISA. Serum
IgG levels are expressed as OD405 nm of a 1/100 serum
dilution. Following 5 days on their respective protocols, mice
were challenged i.n. with influenza virus and were assayed 42 h
later for the shedding of virus in their nasal secretions. Viral titers
are expressed as log10 tissue culture 50% infectious
dose.
