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The Journal of Immunology, 2003, 171: 4700-4707.
Copyright © 2003 by The American Association of Immunologists

TNF-{alpha}-Dependent ICAM-1- and VCAM-1-Mediated Inflammatory Responses Are Delayed in Neonatal Mice Infected with Pneumocystis carinii 1

Mahboob H. Qureshi*,{ddagger}, Joan Cook-Mills§, Dennis E. Doherty{dagger},{ddagger} and Beth A. Garvy2,*,{dagger},{ddagger}

Departments of * Microbiology, Immunology, and Molecular Genetics and {dagger} Internal Medicine, University of Kentucky, and {ddagger} Veterans Administration Medical Center, Lexington, KY 40536; and § Department of Pathology, University of Cincinnati, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neonatal mice have a delayed CD4-mediated inflammatory response to Pneumocystis carinii (PC) infection in the lungs that corresponds to a delayed TNF-{alpha} response and a delayed clearance of the organisms compared with adult mice. Since TNF-{alpha} is known to drive the up-regulation of adhesion molecules, we examined the expression and function of adhesion molecules in the lungs of neonatal mice. The expression of both ICAM-1 and VCAM-1 was significantly lower in the lungs of PC-infected neonatal mice compared with adults. Additionally, migration of neonatal T cells across endothelial cells expressing VCAM-1 and monocyte chemotactic protein-1 was aberrant compared with that in adult T cells, although {alpha}4{beta}1 integrin-mediated adhesion of neonatal lymphocytes was comparable to that of adult lymphocytes. Treatment of neonatal mice with exogenous TNF-{alpha} resulted in increased expression of ICAM-1 and VCAM-1 as well as increased expression of chemokines, resulting in infiltration of CD4+ cells into the lungs. Treatment with exogenous TNF-{alpha} resulted in a trend (although not statistically significant) toward a reduction of PC organisms from the lungs. These data indicate that neonatal lung endothelial cells do not up-regulate ICAM-1 and VCAM-1 in response to PC infection, probably due to depressed TNF-{alpha} production. Additionally, neonatal T cells are defective in the ability to migrate across endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neonates have an inadequate immune response to microbial pathogens, which make them particularly vulnerable to bacterial, viral, and fungal infections (1, 2, 3). It has generally been postulated that the neonatal immune system is immature and therefore does not respond to Ag as efficiently as does the adult immune system (1). In this regard our previous studies reported a delayed resolution of Pneumocystis carinii (PC) 3 organisms in neonatal mice compared with adults (4).

PC is an opportunistic fungal pathogen that often causes significant morbidity and mortality, especially in neonates and infants immunocompromised by AIDS, cancer chemotherapy, or organ transplantation (5, 6, 7). Immunocompetent individuals develop Abs against this organism during early childhood, and thus primary PC pneumonia (PCP) is not common (8). Recent studies have revealed a significant incidence of PC infection in autopsy specimens of children who died from sudden infant death syndrome (9), suggesting that children may frequently carry subclinical PC infection. Despite the availability of effective chemoprophylaxis, PCP in immunocompromised individuals, particularly in neonates, is a common clinical problem (10, 11). Therefore, a better understanding of the neonatal host response mechanism will be useful for developing rational therapeutic strategies to treat neonates against respiratory pathogens, including PC.

Animal experiments and clinical studies indicate that CD4+ T lymphocytes are critical for resolution of PCP (12, 13, 14). We have demonstrated that the recruitment of these cells is delayed in neonates compared with adults, resulting in a delayed resolution of the PC organisms (4, 15). Recently we have reported that neonatal lymphocytes, after adoptive transfer to an adult lung environment, are as efficient as adult lymphocytes in clearing PC organisms (16). Consistent with these results, Adkins et al. (17) have recently reported that neonatal T cells were able to proliferate and up-regulate activation markers when transferred to adult hosts. However, unlike adults, they generated Th2-type responses to keyhole limpet hemocyanin (17). These studies indicate that neonatal lymphocytes function normally in an adult lung environment and support the idea that there is an inadequacy in the neonatal lung environment.

Recruitment of circulatory lymphocytes to the site of inflammation requires the availability of specific activating factors, such as specific cytokines, chemokines, and microbial products (18, 19), resulting in lymphocyte activation, secondary adhesion, and transmigration-chemotaxis (20, 21, 22). Successful extravasation requires the interaction of two or more reciprocal adhesion molecule (lymphocyte receptor-ligand) pairs, such as {alpha}L{beta}2 integrin (CD11a/CD18, LFA-1)/ICAM-1 and {alpha}4{beta}1 integrin (CD49d/CD29,VLA-4)/VCAM-1 on lymphocytes and endothelial cells, respectively (18, 23, 24). Importantly, the {alpha}4{beta}1/VCAM-1 interaction facilitates the recruitment of lymphocytes to inflamed extraintestinal mucosal tissues (18, 24). While the expression of adhesion molecules in adult lungs has been studied under normal and various pathological conditions, such as bronchial asthma (25, 26), chronic obstructive bronchitis (27), and pulmonary infections (28), relatively little is known about the expression of these molecules in neonatal lungs.

In our present studies we have examined the involvement of ICAM-1 and VCAM-1 in cell recruitment to the lungs in response to PC infection. We also examined the differential up-regulation of the endothelial (ICAM-1 and VCAM-1) and lymphocytic ({alpha}L{beta}2 and {alpha}4{beta}1) components of adhesion molecule expression in neonatal and adult lungs as well as the ability of neonatal lymphocytes to migrate through adult endothelial cells in vitro. We further investigated how an alteration in lung environment by exogenous cytokine administration modulates adhesion molecule up-regulation in neonates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Five- to 6-wk-old and mid-term pregnant BALB/c mice were purchased from the National Cancer Institute or Harlan Breeders (Indianapolis, IN). Mice were maintained at the Veterinary Medical Unit of the Veterans Administration Medical Center under specific pathogen-free conditions. C.B-17 SCID mice, used to maintain a source of PC, were also bred at the Veterinary Medical Unit of the Veterans Administration Medical Center in microisolator cages containing sterilized food and water. Protocols for the usage of mice were approved by the Veterans Administration institutional animal care and use committee.

PC infection and TNF-{alpha} administration

Lungs were excised from PC-infected SCID mice and pushed through steel mesh in HBSS. PC was isolated and enumerated by microscopy as previously described (4). Eight-week-old and 48- to 72-h-old BALB/c mice were placed under light halothane anesthesia and inoculated intranasally with 0.5–1 x 106 PC organisms/g of body weight as previously described (4). In selected experiments murine recombinant TNF-{alpha} (BD PharMingen, San Diego, CA) was administered to PC-infected neonates intranasally at 0.025 µg/g of body weight in a 10-µl volume on days 0, 2, 4, and 6 postinfection. Control mice received equal volumes of PBS.

Isolation of lung alveolar, interstitial, and tracheobronchial lymph node (TBLN) cells

Lung and TBLN cells were prepared as described previously (4, 16). Briefly, lungs were lavaged using an intratracheal cannula with five 1-ml washes with cold HBSS/EDTA (3 mM). Right lung lobes were excised, minced, and enzyme-treated at 37°C for 60 min in RPMI containing 3% FCS, 50 U/ml DNase (Sigma-Aldrich, St. Louis, MO), and 1 mg/ml collagenase A (Sigma-Aldrich). Digested lung tissue and lymph node tissues were pushed through mesh screens, and RBC were lysed by treatment with a hypotonic buffer. Cells were resuspended in HBSS for enumeration.

Enumeration of PC

Aliquots of digested lung tissue were diluted, spun onto glass slides, fixed in methanol, and stained with Diff-Quik (Dade International, Miami, FL). PC nuclei were enumerated microscopically by a single investigator, and 20–50 x100 fields were routinely counted (4). The number of PC organisms was expressed as log10 nuclei/right lung lobes. The left lobes were either formalin-fixed or snap-frozen for immunohistochemistry and RNase protection assays, respectively. The limit of detection of PC was log103.6 for both pups and adults.

Preparation of cells for flow cytometric analysis

Cells derived from lungs and TBLN tissue (5 x 105 to 1 x 106 cells) were used for staining with fluorochrome-conjugated Abs purchased from BD PharMingen specific for murine CD4, CD8, CD18, CD19, CD29, CD44, CD49d, and CD62L. Multiparameter analysis was performed using a FACSCalibur cytofluorometer (BD Biosciences, Mountain View, CA). More than 50,000 events were routinely acquired.

Immunohistochemistry

Lung lobes were formalin-fixed, paraffin-embedded, and cut in 7-mm-thick sections that were deparaffinized and hydrated through graded alcohol washes. Lung sections were incubated with rat anti-mouse ICAM-1 (clone KAT-1, Caltag Laboratories, Burlingame, CA) or rat anti-mouse VCAM-1 (clone M/K-2; R&D Systems, Minneapolis, MN) mAbs, followed by biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) and avidin-biotin peroxidase complex (Vector Laboratories). Detection was performed using a 3,3-diaminobenzidine substrate kit (Vector Laboratories). A set of paired sections was stained with isotype-matched control Abs (clone R35-95, rat IgG2a; BD PharMingen). Sections were counterstained with hematoxylin, dehydrated, and mounted. Images were obtained using a Spot digital camera attached to an Eclipse microscope (Nikon, Melville, NY). Adhesion molecule expression was quantitated using image analysis software (Metamorph, Universal Imaging Corp., Downingtown, PA). Data are expressed as the percentage of a thresholded area and include five randomly selected fields per lung section from each individual mouse. The thresholded area is expressed as percentage of the total number of pixels present in a defined region of interest. Saturation, hue, and intensity are the parameters used in this integrated morphometric analysis by Metamorph to set the threshold level. Regions of equal sizes were selected for control and sample images, and the percentage of total pixels was measured using the predetermined parameters set for threshold and expressed as the percent thresholded area.

Lymphocyte adhesion assay

Lymphocyte adhesion assays were performed as previously described (29). A murine high endothelial venule (mHEVa) cell line was grown to confluence in 96-well tissue culture plates (Corning, Corning, NY). Lymphocytes were derived from TBLNs draining PC-infected pup and adult lungs at 10 days postinfection and were treated with calcein acetoxymethyl ester (1 µM) for 10 min at 37°C. Calcein-labeled lymphocytes were added to microwells at 1 x 106/well. To designated wells, anti-{alpha}4 Ab (clone PS-2; BioDesign International, Kennebunk, ME) and isotype-matched rat IgG2b (clone A95-1; BD PharMingen) were added. After incubating for 30 min at 37°C, the plates were read on a microplate fluorometer (Cambridge Technology International, Watertown, MA). The number of adherent lymphocytes per well was calculated using calcein-labeled lymphocyte standard curves generated for each sample.

Lymphocyte migration assay

Lymphocyte migration assays were performed as previously described (29). Briefly, mHEVa cells were plated and grown to confluence in the upper chamber of Transwells with 12-µm pores (Costar, Cambridge, MA). Lymphocytes were derived from TBLNs draining PC-infected pup and adult lungs on day 10 postinfection. The viability of TBLN cells was routinely >95% at the beginning of the assay. Cells were added to the upper chambers on top of the mHEVa monolayers at 7 x 106/well and were incubated at 37°C. To control for confluence of the mHEVa cells, RBC were not eliminated in the lymphocyte preparations. If the cells were confluent, RBCs would not migrate to the lower chambers. For some wells RANTES (PeproTech, Rocky Hill, NJ) was added to the lower chambers at a concentration of 100 or 300 ng/ml. Monocyte chemotactic protein-1 (MCP-1; JE) activity was blocked by addition of 5 µg/ml of specific neutralizing Ab (polyclonal rabbit Ab; eBioscience, San Diego, CA) to the upper and lower chambers 15 min before addition of lymphocytes. An isotype-matched irrelevant rabbit Ab was used as a control and had no activity in the assay (data not shown). Lymphocytes were collected from the lower chambers and enumerated microscopically at the indicated times. Aliquots were stained for flow cytometric analysis using fluorochrome-conjugated Abs specific for CD4, CD8, and CD19 both before and after migration.

Extraction of RNA and RNase protection assay (RPA)

Total RNA was prepared from frozen lung tissue using TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions. Multiprobe DNA templates for chemokines (lymphotactin (Ltn), RANTES, eotaxin, macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1{beta}, MCP-1, MIP-2, and T cell activation gene (TCA)-3), and housekeeping genes (L32 and GAPDH) were purchased from BD PharMingen. RPA was performed using the RiboQuant in vitro transcription and RPA kits (BD PharMingen) according to the manufacturer’s protocol. RNase-protected RNA duplexes were resolved on 5% denaturing polyacrylamide gels. Dried gels were exposed to PhosphorImager screens, and images were developed using a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The intensity of each specific chemokine band was measured using ImageQuant software (Molecular Dynamics). The chemokine mRNA levels were corrected for RNA loaded by dividing the chemokine hybridization signal by the L32 signal for the same sample.

Statistical analysis

The results were tested statistically by Student’s t tests or ANOVA, followed by Student-Newman-Keuls post hoc test where appropriate, using commercially available software (SigmaStat; SPSS, Chicago, IL). Results were determined to be statistically significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of PC clearance and migration of CD4+ T cells into the lungs of mice infected as neonates or adults

Mice were infected as neonates or adults with 2 x 106 or 107 PC organisms, respectively. As shown in Fig. 1, the lung PC burden increased over 3 wk in mice infected as neonates, whereas lung PC burden decreased in adult mice over the same time period. Notably, the recruitment of activated CD4+ T cells into the alveolar spaces was significantly greater in PC-infected adult mice compared with mice infected as neonates (Fig. 1). This is consistent with our previously published data indicating that mice infected with PC as neonates do not mount an inflammatory response until ~3 wk of age and do not clear the infection until 6 wk (4, 15). In contrast, adult mice mount an inflammatory response to PC within days and clear the infection within 3–4 wk. Notably, the inadequate inflammatory response in neonates is not due to a smaller PC inoculum size compared with that in adults, since infection with a 25 times larger dose (5 x 107 organisms) did not influence the kinetics of inflammatory responses in neonates (16).



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  		FIGURE 1. Resolution of lung PC burden and associated CD4+ T cell recruitment are delayed in mice infected as neonates compared with those in adult mice. Lung burdens of PC organisms (symbols) and recruitment of activated (CD44highCD62Llow phenotype) CD4+ cells (bars) were determined by microscopy and FACS analysis, respectively. Data are expressed as log10 PC per right lobe and the proportion of CD4+ T cells present in the alveolar spaces of the same lung lobes. Data are the mean ± SD of three to five mice per group and are representative of three separate experiments. *, p < 0.05 compared with pups at the same time points for both PC burden and CD4+ cell recruitment.

 
Differential up-regulation of ICAM-1 and VCAM-1 expression in pup and adult lungs

CD4+ T cells are critical for efficient PC resolution (12, 13), and we showed that recruitment of these cells into neonatal lungs is delayed compared with that in adults (4, 15). Recruitment of inflammatory cells is largely dependent on appropriate up-regulation of adhesion molecules, including ICAM-1 and VCAM-1 (18, 19, 30). Therefore, we investigated whether the expression of these adhesion molecules is differentially regulated on the endothelium of neonatal and adult lungs. Fig. 2A shows that the expression of ICAM-1 and VCAM-1 is significantly lower in the lungs of mice infected as neonates compared with that in adults at day 14 postinfection. Quantitative analysis of the immunohistochemistry images confirmed that the expression of both ICAM-1 and VCAM-1 was significantly lower in pup lungs compared with adults on days 7, 14, and 21 postinfection (Fig. 2, B and C). The kinetics of adhesion molecule up-regulation corresponded with inflammatory cell infiltration and PC resolution (Fig. 1) (15), suggesting that inadequate ICAM-1 and VCAM-1 up-regulation in neonatal lungs may be involved in the delayed inflammatory response to PC.



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  		FIGURE 2. Up-regulation of ICAM-1 and VCAM-1 expression in response to PC infection is delayed in neonatal lungs compared with that in adult lungs. A, Immunohistochemical staining of pup (a–c) and adult (d–f) lung sections for ICAM-1 (a and d) and VCAM-1 (b and e) on day 14 postinfection. c and f, Staining with isotype-matched control Abs. Magnification, x400. B, Quantitative analysis of immunohistochemical staining for VCAM-1 in the lungs of PC-infected mice at the indicated time points. C, Quantitative analysis of ICAM-1 in the lungs of PC-infected mice at the indicated time points. Data represent the mean ± SD of five fields per lung section from individual mice and three mice in each group. *, p < 0.05 compared with neonatal ICAM-1 or VCAM-1 expression at the same time points.

 
{alpha}4 and {beta}1 integrin expression on pup and adult lymphocytes

Leukocyte recruitment requires interactions between adhesion molecule ligands on the endothelial cells and their receptors on lymphocytes (18, 24). {alpha}4{beta}1 (VLA-4) and {alpha}L{beta}2 (LFA-1) integrins expressed on lymphocytes are receptors for VCAM-1 and ICAM-1, respectively. To determine whether delayed migration of lymphocytes into the lungs of PC-infected neonates was due to inadequate expression of integrins as well as depressed endothelial adhesion molecule expression, we examined the expression of integrin molecules on pup and adult lymphocytes using flow cytometry. Proportions of TBLN-derived CD4+ cells that expressed {alpha}4{beta}1 integrin were not statistically different between pups and adults at 6 days postinfection (Fig. 3). Expression levels, as determined by the mean fluorescence intensity, of {alpha}4 and {beta}1 integrin molecules on CD4+ and CD8+ lymphocytes isolated from draining lymph nodes of PC-infected 6-day-old mice were comparable to those of molecules on adult lymphocytes (data not shown). Moreover, expression levels of CD18 (LFA-1) were similar in pup and adult T lymphocytes isolated from the draining lymph nodes on day 10 postinfection (data not shown). These data indicate that the expression of integrin molecules is comparable in neonatal and adult T cells.



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  		FIGURE 3. Expression of {alpha}4{beta}1 integrin molecules on neonatal CD4+ T cells is comparable with that on adult cells on day 6 postinfection. TBLN-derived lymphocytes were analyzed by FACS for determining the proportion of CD4+ cells that express {alpha}4{beta}1 integrin molecules on their surface. Representative scatter plots showing {alpha}4 and {beta}1 expression on CD4+ cells are shown in the upper panels. The percentage of CD4+ cells that express both {alpha}4 and {beta}1 is shown. Quadrants were set using CD4+ cells stained with isotype-matched control Abs such that background staining in the upper and lower right quadrants is <1% of the total events shown in the lower panel. Data are representative of four or five samples from separate mice and are consistent over days 6, 10, and 14 postinfection.

 
VCAM-1-mediated lymphocyte adhesion to endothelial cells

Firm adhesion of lymphocytes to the endothelial surface is an essential step before successful extravasation of these cells to the sites of inflammation (19, 23). To determine whether the integrins expressed on neonatal lymphocytes mediate firm adhesion, we examined the ability of neonatal and adult lymphocytes to adhere to an endothelial cell line (mHEVa) constitutively expressing VCAM-1 and a novel ligand for {alpha}4{beta}1 (31). Lymphocytes were collected from the TBLN draining PC-infected pup and adult lungs on day 7 postinfection and were incubated on mHEVa cells. As shown in Fig. 4, there was no significant difference in the total numbers of pup and adult lymphocytes adherent to endothelial cells after incubation for 30 min. Interestingly, adhesion of both pup and adult lymphocytes was inhibited in the presence of anti-{alpha}4 Ab compared with isotype-matched Ab (data not shown). These data suggest that neonatal lymphocytes from infected mice are competent to perform the initial step of cell adhesion before extravasation.



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  		FIGURE 4. Adhesion of neonatal and adult lymphocytes to endothelial cells is comparable. TBLN-derived lymphocytes were cultured on mHEVa endothelial cell monolayers. The numbers of adherent lymphocytes per well were calculated using calcein-labeled lymphocyte standard curves generated for lymphocytes from each sample. Data are the mean ± SD of three wells per group, each well representing an individual pool of two to seven mice, and are representative of two separate experiments.

 
Differential in vitro migration of pup and adult lymphocytes through endothelial cells

Integrin-mediated strong adhesion to the endothelium is followed by migration of lymphocytes through endothelial cell junctions to the site of infection/inflammation (18, 19, 23). Therefore, to address the next step of cell recruitment, we examined whether migration of pup lymphocytes through endothelial cell monolayers is as efficient as migration of adult lymphocytes. The mHEVa cell line provides a model of VCAM-1-mediated adhesion and migration. Moreover, these cells produce MCP-1, but not Ltn, RANTES, eotaxin, MIP-1{alpha}, MIP-1{beta}, MIP-2, or TCA-3, as determined by RPA and ELISA (data not shown). Lymphocytes isolated on day 7 postinfection from TBLNs of mice infected with PC as neonates or adults were allowed to migrate through endothelial cell monolayers in a Transwell culture system over a 48-h period. The total number and proportions of pup TBLN cells that migrated through mHEVa cell monolayers was significantly lower than those in adults (Fig. 5, A and C). In addition, the proportions of pup TBLN-derived CD4+ and CD8+ T cells and CD19+ B cells that migrated through the mHEVa cell monolayer were significantly lower compared with those in adults (Fig. 5B).



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  		FIGURE 5. Neonatal and adult lymphocytes migrate differentially through endothelial cells. A, TBLN-derived lymphocytes were cultured in the upper chamber of Transwells with confluent monolayers of mHEVa cells. Migration of neonatal lymphocytes through mHEVa cell monolayer was determined after 24 and 48 h of culture, and data are expressed as the cumulative number of cells migrated. B, Proportions of neonatal and adult CD4+ and CD8+ T cell and B cells that migrated through endothelial cells. Data are expressed as the percentage of total cells of each phenotype used to begin the culture. C, Proportions of neonatal and adult lymphocytes that migrated through endothelial cell. Cells were enumerated by microscopy after 24 h of culture. Data represent the mean ± SD of six wells per group. Each well represents an individual pool of two to seven mice. Data are representative of two separate experiments. *, p < 0.05 compared with the neonatal lymphocytes at the same time points.

 
To determine whether adult lymphocytes migrated in response to MCP-1, a neutralizing Ab was placed in both chambers of the cultures. Fig. 6 shows that anti-MCP-1 Ab significantly inhibited the migration of adult lymphocytes, but had no effect on neonatal lymphocytes. Moreover, addition of another T cell chemokine, RANTES, to the cultures caused a 2-fold increase in the number of pup lymphocytes that migrated through the mHEVa cells; however, this was not a statistically significant increase (Fig. 6). RANTES did not affect the migration of adult lymphocytes, suggesting that maximal VCAM-1-mediated migration occurred in response to MCP-1.



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  		FIGURE 6. Neonatal lymphocytes fail to migrate in the presence of either MCP-1 or RANTES. TBLN-derived lymphocytes were cultured in the upper chamber of Transwells with confluent monolayers of mHEVa cells. The migration of neonatal lymphocytes through mHEVa cell monolayers was determined after 24 h of culture, and data are expressed as the cumulative number of cells migrated. Data represent the mean ± SD of three wells per group. Cells from neonates (n = 16) and adults (n = 4) were pooled to obtain enough cells for all experimental groups. *, p < 0.05 compared with adults receiving the same treatment; +, p < 0.05 compared with adults with isotype-matched Ab.

 
Adhesion molecule and chemokine up-regulation after exogenous TNF-{alpha} administration is associated with CD4+ T cell migration into neonatal lungs

TNF-{alpha} is a proinflammatory cytokine with a wide range of effects on inflammatory responses, including up-regulation of chemokines and adhesion molecules (32, 33). We have reported that up-regulation of TNF-{alpha} mRNA is delayed in neonatal lungs in response to PC infection (16). Therefore, we examined whether changing the cytokine milieu in the neonatal lung environment would affect the migration of inflammatory cells into the lungs. PC-infected neonates were given intranasal inoculations of recombinant murine TNF-{alpha} or PBS on days 0, 2, 4, and 6 postinfection. Immunohistochemical analysis of formalin-fixed lung tissue from these animals on day 7 postinfection revealed that TNF-{alpha} treatment induced enhanced expression of ICAM-1 and VCAM-1 on neonatal lung endothelium compared with PBS treatment (not shown). Fig. 7 represents quantitative analysis of the immunohistochemical images and confirms that there is a significant difference in the expression of ICAM-1 and VCAM-1 between the TNF-{alpha}-treated mice and control neonatal mice.



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  		FIGURE 7. The expression of ICAM-1 and VCAM-1 molecules is up-regulated in neonatal lungs upon exogenous TNF-{alpha} administration. Neonatal mice were infected with PC and treated intranasally with exogenous TNF-{alpha} at 0.025 µg/g of body weight or with PBS on days 0, 2, 4, and 6 postinfection. Lungs were fixed on day 7 postinfection. Quantitative analysis of immunohistochemical staining for ICAM-1 and VCAM-1 in TNF-{alpha}- and PBS-treated lungs on day 7 postinfection is shown. Nonspecific background staining using isotype-matched Abs is shown as bars labeled control. Data represent the mean ± SD of five fields per lung section from individual mice and three mice in each group. *, p < 0.05 compared with PBS-treated mice at the same time points.

 
Treatment of neonates with exogenous TNF-{alpha} resulted in a significant increase in the expression of Ltn, RANTES, MCP-1, MIP-1{alpha}, MIP-1{beta}, MIP-2, and TCA-3 on day 7 postinfection (Fig. 8). However, by day 14 postinfection, differences in Ltn, RANTES, MIP-1{alpha}, MIP-1{beta}, and MCP-1 expression between the TNF-{alpha}-treated and control groups had disappeared (Fig. 8). The expression levels of Ltn, MIP-1{alpha}, and MCP-1 mRNA did not increase further after cessation of TNF-{alpha} administration on day 6 postinfection. By that time the neonates treated with PBS had started to up-regulate chemokine expression in response to PC infection, and both groups had comparable expression of chemokine mRNA by day 14 postinfection (Fig. 8). Interestingly, the expression of MCP-1 was decreased almost to the basal level after cessation of TNF-{alpha} administration. These data indicate that exogenous TNF-{alpha} stimulates the up-regulation of chemokine mRNA expression in PC-infected neonates.



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  		FIGURE 8. The expression of chemokine mRNA is up-regulated in neonatal lungs after exogenous TNF-{alpha} administration. Quantitative analysis of the RPA images showing Ltn, RANTES, MIP-1{alpha}, and MCP-1 mRNA expression in the lungs of PC-infected neonates after TNF-{alpha} or PBS treatment as described in Fig. 7. Neonatal mice were infected with PC and treated with TNF-{alpha} or PBS. Data are the mean ± SD of three or four mice per group and are representative of two separate experiments. *, p < 0.05 compared with the PBS-treated mice at the same time points.

 
In addition to up-regulation of chemokine and endothelial cell adhesion molecule expression, TNF-{alpha} stimulated a significant increase in the numbers of activated CD4+ T cells(CD44highCD62Llow phenotype) into the lymph nodes, alveolar spaces, and lung interstitium as early as day 9 postinfection (Fig. 9). The mean number of activated CD4+ cells was always higher in the TBLNs of cytokine-treated pups, even though it was not statistically significant on days 16 and 25 postinfection (Fig. 9). Recruitment of activated CD4+ T cells into the alveolar spaces and lung interstitium of TNF-{alpha}-treated mice was significantly increased on day 16 and on days 16 and 25 postinfection, respectively (Fig. 9). Even though recruitment of activated CD4+ cells was significantly increased after TNF-{alpha} treatment, resolution of PC organisms was not significantly enhanced in the cytokine-treated mice over time (data not shown). However, on day 25 postinfection, the lung PC burden was 5-fold lower in TNF-{alpha}-treated lungs compared with PBS-treated lungs (log10, 6.12 ± 0.48 compared with 6.83 ± 0.41, respectively). This represents a trend toward clearance in the TNF-{alpha}-treated group.



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  		FIGURE 9. Exogenous TNF-{alpha} administration enhanced the recruitment and activation of CD4+ T cells in the neonates. Neonatal mice were infected with PC and treated with TNF-{alpha} or PBS as described in Fig. 7. FACS analysis determined the total number of activated (CD44highCD62Llow phenotype) CD4+ cells in lymph nodes, alveolar spaces, and lung interstitium at the indicated time points. Data are the mean ± SD of three or four mice per group and are representative of two separate experiments. *, p < 0.05 compared with the PBS-treated mice at the same time points.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The underlying mechanism of delayed clearance of PC organisms from neonatal lungs is not well understood. We have demonstrated that up-regulation of cytokines and chemokines as well as CD4+ cellular infiltration into the lungs in response to PC infection is delayed in neonatal mice (15, 16). We have also demonstrated that the inadequacy of the neonatal lung immune response is largely due to the lung environment rather than an immature lymphocyte population (17, 18). However, precisely which component of the neonatal lung environment is insufficient has yet to be determined. Our results described here suggest that delayed up-regulation of ICAM-1 and VCAM-1 adhesion molecules in response to PC infection is involved in the sluggish recruitment of inflammatory cells into neonatal lungs.

VCAM-1 has been shown to be important in protective immunity to pulmonary infections other than PC, such as Mycobacterium tuberculosis (34). However, little is known regarding the roles of ICAM-1 and VCAM-1 in defense of neonatal lung infections. In the present studies we have demonstrated that neonates have a reduced level of expression of ICAM-1 and VCAM-1 in their lungs in response to PC infection compared with adults. This reduced adhesion molecule expression probably contributes to the inefficient cellular recruitment into neonatal lungs in response to PC infection. We have previously reported that T cells isolated from the draining lymph nodes of PC-infected pups proliferate and produce IFN-{gamma} in response to anti-CD3; however, T cells do not appear in the lungs of PC-infected mice until ~3 wk postinfection (16). To determine whether neonatal cells could also migrate through endothelial cells, we used a model of VCAM-1-mediated migration (31). We observed that adhesion of neonatal lymphocytes to an endothelial cell line was VCAM-1 dependent and comparable to that in adults. Thus, neonatal lymphocytes have the potential to adhere to endothelial cells via adhesion molecules.

Even though ex vivo adhesion of neonatal lymphocytes was comparable to that of adult cells, migration of neonatal lymphocytes through the mHEVa cell line that expresses VCAM-1 and MCP-1 was significantly less efficient (Figs. 5 and 6). Almost 5-fold more adult TBLN cells migrated through the mHEVa cell layer than did pup cells. This suggests that after adhesion, neonatal lymphocytes are not signaled to migrate. Transendothelial cell migration is regulated by a series of intracellular signaling events that involves calcium mobilization, protein kinase C phosphorylation, and redistribution and rearrangement of cytoskeletal proteins (35, 36). Therefore, the lack of sufficient intracellular signaling in the neonatal lymphocytes may interfere with efficient transendothelial migration. Recent studies have reported that transendothelial migration of leukocytes is regulated by integrin-mediated, calcium-dependent, cytoskeletal organization (37, 38). Therefore, a defect in calcium mobilization in neonatal lymphocytes may not preclude efficient adhesion to endothelial cells.

Consistent with our results, it has been reported that a mutant Jurkat T cell line that expresses {alpha}4{beta}1 integrin but is defective in inducing {alpha}4{beta}1 integrin activation was able to adhere to endothelial cells, but was inefficient at transendothelial migration (39). This indicates that lymphocyte migration, but not adhesion to endothelial cells, is dependent on {alpha}4{beta}1 integrin activation. Further, adhesion was blocked with anti-{alpha}4 Ab, indicating a critical role of {alpha}4{beta}1 integrin and VCAM-1 interaction for cell adhesion (39). In our system neonatal lymphocytes expressed {alpha}4 integrin molecules required for adhesion, but it is possible that they were defective in inducing {alpha}4 integrin outside-in signals that were critical for transendothelial migration. In this regard it has also been shown using our in vitro system that pretreatment of mouse splenic lymphocytes with irreversible inhibitors of calmodulin or phosphotidylinositol 3-kinase significantly decreased lymphocyte migration through the mHEVa cell monolayers (29). Interestingly, lymphocytic adhesion to endothelial cells was not decreased by these inhibitors, suggesting that, subsequent to the initial cell adhesion event, these signaling molecules in lymphocytes were required for migration (29). Thus, neonatal lymphocytes may be deficient in integrin-stimulated calmodulin- and phosphotidylinositol 3-kinase-mediated signaling, resulting in less efficient migration.

Alternatively, as has been reported in human neonatal leukocytes, rearrangement of cytoskeleton may be defective in neonatal mouse lymphocytes (40, 41). Defective migration of neonatal lymphocytes is likely to be multifactorial and warrants further investigation. Studies are underway in our laboratory examining the intracellular signaling events and reorganization of actin filaments in neonatal T lymphocytes in response to ligation of integrin molecules.

The resolution of PCP in immunocompetent adult mice is associated with the up-regulation of proinflammatory cytokines (IFN-{gamma} and TNF-{alpha}), chemokines (Ltn, RANTES, MIP-1{alpha}, MIP-1{beta}, and MCP-1), and adhesion molecules (ICAM-1 and VCAM-1) with subsequent recruitment of activated CD4+ T lymphocytes (15, 16, 42, 43, 44). As opposed to adults, neonates have a delayed up-regulation of lung cytokines (IFN-{gamma} and TNF-{alpha}) and chemokines (Ltn, RANTES, MIP-1{alpha}, MIP-1{beta}, and MCP-1) in response to PC infection (15, 16). In our present study we modulated the neonatal lung environment by direct administration of exogenous TNF-{alpha} into their lungs. An important role for TNF-{alpha} in chemokine and adhesion molecule up-regulation in adults has been reported in several studies (32, 33, 45). The significant up-regulation of chemokine and adhesion molecule mRNA along with enhanced activated CD4+ cell recruitment into neonatal lungs after TNF-{alpha} treatment indicate that insufficient TNF-{alpha} production in response to PC plays a major role in the delayed inflammatory response in neonates. Moreover, the loss of enhanced expression of chemokines 1 wk after cessation of TNF-{alpha} treatment suggests that sustained chemokine up-regulation is dependent on the availability of TNF-{alpha} in the local environment.

Notably, MCP-1 up-regulation was reduced to almost basal levels after cessation of TNF-{alpha} administration. MCP-1 has been reported to be critical for CD4+ T cell recruitment and induction of protective immune responses against Cryptococcus neoformans infection (46). Interestingly, in our in vitro migration assay the presence of MCP-1 or RANTES did not stimulate the migration of neonatal lymphocytes to any significant degree compared with adult cells. This inability to migrate in the presence of chemokines was consistent with the kinetics of migration of activated CD4+ cells into the alveolar spaces. Even though TNF-{alpha}-driven chemokine mRNA expression was increased within 1 wk of infection, there were not appreciable numbers of infiltrating CD4+ cells into the lungs until 2 wk postinfection. This suggests that neonatal T cells either do not express adequate chemokine receptors or that signaling through the receptors is defective. We are actively investigating both these possibilities.

Exogenous treatment of neonatal mice with TNF-{alpha} resulted in increased expression of lung chemokines and expedited infiltration of CD4+ cells with an activated phenotype into the lungs. Despite this, the lung PC burden was consistently decreased compared with that in vehicle controls, but not enough to reach statistical significance (data not shown). One possibility is that TNF-{alpha} treatment resulted in nonspecific recruitment of T cells into the lungs. Since there was a trend toward reduction in PC, it is also possible that we simply did not carry the experiments out far enough to observe clearance. We favor this explanation because even in the presence of exogenous TNF-{alpha} during the first week postinfection, there were still not appreciable numbers of T cells in the alveoli until day 16, and this increased further (~3-fold) over the next 10 days. We would expect that considerable clearance of the organisms would take place after day 25, when we found a burst of infiltrating T cells in the alveoli.

Taken together, these studies indicate that up-regulation of ICAM-1 and VCAM-1 expression appears to be inadequate for generating an immune response in neonatal lungs compared with adults. Neonatal lymphocytes are competent in expressing {alpha}4{beta}1 integrin receptors and binding their ligand, VCAM-1. However, subsequent to adhesion, decreased migration of neonatal lymphocytes through endothelial cell monolayers suggests that neonatal lymphocytes are deficient in inducing the proper signaling required for cell migration. Consistent with our previous studies in which deficient TNF-{alpha} up-regulation in neonatal lungs in response to PC was associated with delayed cellular responses (16), we demonstrate that modulating the neonatal lung environment by direct administration of TNF-{alpha} into the lungs results in enhanced adhesion molecule and chemokine expression and cellular recruitment. Thus, our data clearly indicate that the neonatal lung environment is inadequate to support the induction of an effective immune response. In addition, the migration efficiency of neonatal lymphocytes is lower than that of adults. Determining the mechanism for lack of TNF-{alpha} up-regulation in neonatal lungs in response to PC is of significant interest. Understanding the nature of the unresponsiveness in neonatal lungs should be useful in developing strategies for protecting neonates from pathogenic fungi, including PC.


   Acknowledgments
 
We thank Kevin Schuer, Wayne Young, and Melissa Hollifield for expert technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HL62053 and HL64524 (to B.G.) and R01HL68171 (to J.C.-M.) and American Lung Association Fellowship Grant RT-052-N (to M.Q.). Back

2 Address correspondence and reprint requests to Dr. Beth A. Garvy, Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky Medical Center, Room MN668, 800 Rose Street, Lexington, KY 40535. E-mail address: bgarv0{at}uky.edu Back

3 Abbreviations used in this paper: PC, Pneumocystis carinii; Ltn, lymphotactin; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; PCP, Pneumocystis carinii pneumonia; RPA, RNase protection assay; TBLN, tracheobroncheal lymph node; TCA, T cell activation gene. Back

Received for publication April 4, 2003. Accepted for publication August 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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