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Originally published online as doi:10.1189/jlb.1206729 on March 16, 2007

Published online before print March 16, 2007
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(Journal of Leukocyte Biology. 2007;81:1352-1361.)
© 2007 by Society for Leukocyte Biology

Deficient inflammatory response to UV radiation in neonatal mice

Agnieszka Wolnicka-Glubisz*,1, Jesse Damsker{dagger}, Stephanie Constant{dagger}, Stephanie Corn{ddagger}, Edward De Fabo* and Frances Noonan*,2

* Laboratory of Photobiology and Photoimmunology, Department of Environmental and Occupational Health, School of Public Health and Health Services, and
{dagger} Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University Medical Center, Washington, DC, USA; and
{ddagger} Mouse Phenotyping Shared Resource, Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, USA

2 Correspondence: Laboratory of Photobiology and Photoimmunology, Department of Environmental and Occupational Health, School of Public Health and Health Services, George Washington University Medical Center, Ross Hall, 2300 Eye Street, N.W., Washington, DC 20037, USA. E-mail: drmfpn{at}gwumc.edu

ABSTRACT

Mechanisms of juvenile susceptibility to cancer are not well understood. The immune response in neonates favors nonresponsiveness or TH2-dominant responses, raising the question of a role for neonatal immunity in this susceptibility. We have investigated the postulate that the inflammatory response differs in neonatal and adult skin. We found no inflammatory infiltrate into neonatal mouse skin in response to UV irradiation as a function of time, dose, or wavelength, although UV-induced DNA damage was readily detected. In contrast, UV irradiation of adult mice initiated a dose- and time-dependent influx of inflammatory cells, chiefly CD11b+Ly6G+ neutrophils, into the skin, detected by immunohistochemistry and quantitated by FACS analysis. This inflammatory response was initiated by UVB (290–320 nm) but not by UVA (320–400 nm). Further, in neonates, in contrast to adults, neither topical trinitrochlorobenzene (TNCB) nor i.p. thioglycollate initiated an inflammatory infiltrate. Conversely, topical TNCB applied to neonates was tolerogenic, resulting in a subsequent antigen-specific decrease of the contact-hypersensitivity response in adults. Neonatal blood contained abundant neutrophils, which exhibited impaired chemotaxis to the chemokine growth-related oncogene-{alpha} but efficient chemotaxis to the bacterial product fMLP, concomitant with decreased expression of CXCR2 but normal levels of CD11b. We propose this neonatal deficiency in the inflammatory response is a significant, previously unrecognized factor in neonatal immune tolerance and may contribute to neonatal susceptibility to cancer, including melanoma and other UV-induced cancers.

Key Words: ultraviolet • inflammation • neutrophil • contact hypersensitivity

INTRODUCTION

Neonates appear to have an increased sensitivity to carcinogenesis [1 ], but the mechanisms are defined incompletely. For example, epidemiologic studies indicate that childhood sunburn is a major risk factor for subsequent development of cutaneous malignant melanoma, but the basis for this juvenile susceptibility is not understood [2 , 3 ].

It is known that neonatal mice show a propensity to the development of TH2 responses and immune tolerance [4 , 5 ]. Although it is clear that under certain conditions, neonatal mice can mount mature responses [6 , 7 ], there is an accumulation of observations indicating quantitative and qualitative differences in neonatal adaptive immunity. The reasons are complex (reviewed in ref. [4 ]) and reflect neonatal, environmental influences as well as the intrinsic capabilities of neonatal cells to respond. Thus, the nature of the neonatal, inflammatory response to carcinogens, such as UV radiation, is of considerable interest in the context of these altered neonatal immune responses and cancer susceptibility.

There is considerable evidence that chronic inflammation is cancer-promoting through production of angiogenic, growth-promoting, and immunosuppressive factors [8 , 9 ]. In experimental epithelial skin cancers, the role of inflammation in response to tumor promoters (reviewed in ref. [10 ]) and in response to UV radiation [11 ] is well described. For the UV induction of cutaneous malignant melanoma, however, the question of the role of inflammation has, until the recent development of appropriate animal models, been difficult to address.

We have previously derived a model for UV-induced melanoma using the hepatocyte growth factor/scatter factor (HGF/SF)-transgenic mouse, in which neonatal, but not adult, exposure to UV initiates melanomas, which recapitulate the histopathology of human disease [12 13 14 ]. The UVB doses used to initiate melanoma in this model were predicted [12 ] to cause significant erythema or sunburn, a cutaneous response marked by inflammation [15 , 16 ]. As UV irradiation is effective only in neonates in the HGF/SF melanoma model, this raises the question of the nature of the inflammatory response in neonatal animals [17 ].

We have therefore investigated the inflammatory response to UV radiation and other agents in neonatal and adult FVB wild-type and melanoma-prone, HGF/SF-transgenic mice. We have identified a profound defect in the infiltration of inflammatory cells in wild-type and transgenic neonates. We postulate this inflammatory defect is a hitherto unrecognized contributor to neonatal cutaneous-immune tolerance and in susceptible animals, may contribute to neonatal susceptibility to carcinogenesis.

MATERIALS AND METHODS

Animals
Inbred albino FVB/NCr strain mice were obtained at 4–6 weeks of age from Frederick Cancer Research Facility (Frederick, MD, USA). Transgenic HGF/SF mice, in which expression of a murine HGF/SF cDNA is driven by the metallothionein promoter and locus control regions [18 ], were supplied courtesy of Dr. Glenn Merlino [National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD, USA] and were maintained on the FVB/NCr genetic background. Neonatal and adult mice were produced in the Animal Facility of the George Washington University Medical Center (Washington, DC, USA). All animal work was performed in accordance with guidelines established by the NIH Guidelines for Humane Care and Use of Laboratory Animals. DNA was isolated from mouse tail clip using DNeasy tissue kit (Qiagen, Valencia, CA, USA) and genotyped for the HGF/SF transgene as described [19 ].

UV irradiation
Neonatal pups (3–5 days of age) or adult (20–25 days of age) FVB wild-type and HGF/SF-transgenic mice were irradiated under identical conditions with a bank of F40 sunlamps using methods described previously. Adult mice were shaved and immobilized prior to irradiation [20 ]. Isolated UVB (280–320 nm) or UVA (320–400 nm) wavebands were produced with bandpass filters in combination with a 2.5-kW xenon lamp, and mice were irradiated as described [12 ].

Edema
Skin-fold thickness was measured with a spring-loaded micrometer (Mitutoyo 7309, Tokyo, Japan) at five sites on adults and three sites on neonates, anterior to posterior, along the midline of the back. Four to six independent experiments were performed on littermates. There were no significant differences in body weight between unirradiated and UV-irradiated pups within a litter.

Contact hypersensitivity (CHS)
A single application of the contact sensitizer TNCB (2,4,6-trinitrochlorobenzene; Polysciences Inc., Warrington, PA, USA) was used to induce inflammation and contact sensitivity. A topical application of a 1% TNCB solution in acetone to dorsal or abdominal skin, after shaving in adults (100 µl per adult or 25 µl per pup), was used. Contact sensitivity was elicited and measured by ear swelling after rechallenge on the ears with 1% TNCB as described [20 ]. Some adult animals were sensitized and challenged with 1% OX (4-ethoxymethlene-2-phenyl-2-oxazolin-5-one; Sigma Chemical Co., St. Louis, MO, USA) in ethanol and ear swelling determined as for TNCB.

Peritoneal inflammation
Pups and adult mice were injected i.p. with sterile thioglycollate (TG; 3% wt/vol in water, 50 ml/kg animal). The effects were monitored 4 h and 24 h after TG injection [21 ]. Peritoneal cells were harvested by i.p. lavage using ice-cold PBS.

Preparation of single cell suspensions from skin
Preparations of disaggregated skin cells were prepared as described [22 ]. In brief, dorsal skin was disinfected with 70% ethanol, and blood vessels and s.c. tissue were removed. Skin samples were digested with liberase (0.5 mg/ml liberase in DMEM, Roche Diagnostics, Nutley, NJ, USA) and homogenized using a Medimachine (DakoCytomation, Denmark). Cells were washed with PBS supplemented with 0.025% sodium azide and 1% BSA (Sigma Chemical Co.).

Antibodies and FACS analysis
In all experiments, cells (3x106cells/ml) were preincubated in 5% mouse serum (Caltag, S. San Francisco, CA, USA) in PBS with rat antimouse CD16/CD32 antibody (1:100, BD PharMingen, San Diego, CA, USA) to block Fc{gamma}RIII/II. Then, samples of 6 x 105 cells were incubated with appropriate mAb for a further 30 min on ice in the dark. The following mAb were used at dilutions indicated: PE rat antimouse-Ly6G (BD Biosciences, San Jose, CA, USA, 1:200), FITC rat antimouse-MHC Class II (BD PharMingen, 1:40), PE rat antimouse-F4/80 (Caltag, 1:200), and APC or PE rat antimouse-CD11b (Caltag, 1:600). Isotype-matched control antibodies, rat antimouse-IgG2a (Caltag), or rat antimouse-IgG2b (Caltag), conjugated with appropriate fluorophore, were used at the same dilution as the primary antibodies. PE-conjugated antibody to CXCR2 (R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer’s instructions. Cells were washed twice with PBS supplemented with 1% BSA and 0.0025% sodium azide and analyzed using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Cell sorting was done using a FACSAria (Becton Dickinson). Nonviable cells were gated out of profiles using propidium iodide (Sigma Chemical Co.). At least 10,000 cells were analyzed using the CellQuest program.

Chemotaxis assays
Circulating neutrophils were enriched from mouse peripheral blood collected from three to five adult mice and 20–25 neonates. We were unable to obtain simultaneously a sufficient number of age-matched, neonatal HGF/SF transgenics for this assay. Blood samples were lysed to remove RBC, and Gr-1+ cells were prepared by MACS separation using a positive selection kit (Miltenyi Biotec, Aurora, CA, USA). Chemotaxis assays were set up as described [23 ] using as chemoattractant growth-related oncogene (Gro)-{alpha} (ProSpec-Tany TechnoGene Ltd., Israel, concentration optimized to 10 ng/ml) or bacterial peptide analog fMLP (Sigma Chemical Co., concentration optimized to 10–6M).

Immunohistochemistry for DNA damage
Sections (5 µm) of formalin-fixed, paraffin-embedded skin were rehydrated, trypsinized (0.125%, Mediatech, Washington, DC, USA) for 30 min at 37°C, and washed with PBS and distilled water. Endogenous peroxidase was blocked by incubation in freshly prepared 3% H2O2 in methanol for 30 min at room temperature. Slides were treated with 1 N HCl for 30 min at room temperature, washed with dH2O and PBS, blocked with 5% goat serum in PBS for 15 min at room temperature, and then incubated with a HRP-conjugate antithymine dimer mAb (1:50 in 0.1% BSA, Kamiya, Seattle, WA, USA) overnight at 4°C. Sections were rinsed with PBS and developed with diaminobenzidine (DAB) using a Vector-DAB kit (Vector Laboratories, Burlingame, CA, USA) for 5 min and counterstained with hematoxylin.

Immunofluorescence for CD11b
Frozen sections (10 µm) were fixed for 10 min with acetone and air-dried overnight. Sections were rinsed with PBS, blocked for 30 min with 2% rat serum (Caltag), and stained for 1 h with rat antimouse CD11b-FITC antibody (1:20, Caltag) in 0.1% BSA in PBS. Negative controls were stained with isotype control (FITC-Ig2Ga, Caltag).

Statistical analysis
Significance of differences was tested using the Student’s t test. Results are expressed as mean value ± SEM.

RESULTS

Neonatal mouse skin responded to UV radiation with edema but no inflammatory infiltrate
Irradiation of adult mice with F40 sunlamps resulted in marked inflammatory infiltrate in the upper dermis and epidermis of adult skin (Fig. 1A ), which was similar in FVB-HGF/SF transgenics and FVB wild-type animals (Supplemental Fig. 1). In contrast, there was no observable infiltrate in neonatal skin of either genotype (Fig. 1A and Supplemental Fig. 1). Erythema was readily visible in adults but not in neonates (not shown), and increased vascularity was readily visible in adult skin 48 h after UV but was not detectable in neonates (Fig. 1C) . Significant edema, determined by skin-fold thickness, was, however, detected in adult and neonatal skin, indicating blood vessel leakage in response to UV. Skin thickness (cmx10–3±SEM) for unirradiated and UV-treated littermates was, for unirradiated adults, 75.5 ± 4.3, and 48 h after UV, 120 ± 18.3 (P<0.001), and for unirradiated neonates, 109 ± 5.1, and 48 h after UV, 124 ± 2.9 (P<0.002). This finding was replicated three times with four to six mice per group.


Figure 1
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  		Figure 1. UV irradiation initiates DNA damage but not inflammatory infiltrate or vascularization in neonatal skin. (A) H&E stain of HGF/SF-transgenic FVB mouse skin 48 h after irradiation with 8.4 kJ/m2 UV from an F40 sunlamp (for spectral output, see Supplementary Fig. 3). Infiltrate is present in adult but not neonatal skin. Original bars represent 0.1 mm. (B) DNA damage (brown nuclear staining) detected in adult and neonatal skin with a HRP-conjugated, antithymine dimer antibody 1 h after UV irradiation as for A. Observations were similar in HGF/SF-transgenic and in wild-type FVB strain animals (see Supplemental Fig. 2). Insets, lower right, represent higher magnification. (C) Underside of unirradiated or irradiated skin 48 h after UV, showing increased vascularity in adult but not neonatal skin. Edema was observed in adult and neonatal skin in response to UV (see text).

 
UV-induced DNA damage was similar in adult and neonatal skin
Neonatal mouse skin was significantly thicker than adult skin, as demonstrated in the skin-fold thickness determinations above. In particular, neonatal skin had a thicker epidermis (five to six cell layers vs. two to three for adults) and also a thicker stratum corneum (Fig. 1A and 1B) . To exclude the possibility of screening of inflammatory targets from UV radiation in neonatal skin, DNA damage was visualized by immunohistochemistry using an antibody to cyclobutane pyrimidine dimers. UV irradiation induced DNA damage in adult and neonatal animals in HGF/SF transgenics and in wild-type skin (Fig. 1B and Supplemental Fig. 2). Thus, the lack of inflammatory infiltrate could not be explained by a lack of UV penetration into neonatal skin.

Inflammatory CD11b+Ly6G+ cells infiltrated adult skin after UV radiation
Immunohistochemistry of adult and neonatal mouse skin indicated resident CD11b+ cells in unirradiated, adult skin, which were present sparsely in neonatal skin (Fig. 2A , left panels). After UV irradiation with F40 sunlamps, an influx of CD11b+ cells was observed in adult skin but was not detectable in neonatal skin (Fig. 2A , right panels). This was confirmed by quantifying the infiltrate by FACS analysis of disaggregated skin cells (Fig. 2B) . The percentage of CD11b+ cells present in disaggregated, untreated adult skin determined by FACS analysis (16.08%±2.84) was significantly higher than in neonatal skin (8.6%±1.27, P<0.0001). Double-staining of adult skin cells indicated resident CD11b+ cells to be negative for the neutrophil marker Ly6G. (Fig. 2C , left panel). In UV-irradiated skin, however, there was an additional population of CD11b+Ly6G+-infiltrating cells (Fig. 2C , right panel). FACS sorting and Giemsa staining of these cell populations confirmed that in UV-irradiated adult skin, CD11b+Ly6G– cells were a heterogeneous population of macrophages, lymphocytes, eosinophils, and large mononuclear cells (Fig. 2D , left panel), whereas CD11b+Ly6G + cells were almost exclusively neutrophils (Fig. 2D , right panel). Infiltrating inflammatory cells were quantitated in skin cell preparations by FACS analysis as a function of time after UV irradiation using Ly6G and CD11b as markers. In addition to CD11b+ and Ly6G+ cells, the percentage of MHC Class II+ cells [chiefly epidermal Langerhans cells (LC)] was also determined. Dose- and wavelength-dependent decreases in epidermal LC as a function of UV irradiation have been well described [24 , 25 ]. UV irradiation induced a significant increase in CD11b+ cells (Fig. 2E , left panel) and in Ly6G+ cells (Fig. 2E , center panel) and a decrease in MHC Class II+ cells (Fig. 2E , right panel) in adult mouse skin, 24–72 h after irradiation with F40 sunlamps. In contrast, no significant changes were detected in any of the three cell populations in neonatal skin (Fig. 2E , all panels) at any of the time-points.


Figure 2
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  		Figure 2. UV irradiation induces infiltration of CD11b+Ly6G+ cells in adult but not in neonatal mouse skin. (A) CD11b+ cells (green, upper right panel) infiltrate adult but not neonatal FVB mouse skin 48 h after UV irradiation with F40 sunlamps as for Figure 1 . Original bar represents 0.1 mm. (B) FACS analysis of disaggregated skin cells from adults and neonates confirms adult CD11b+ infiltrate (circled). (C) FACS analysis of unirradiated (left panel) and UV-irradiated (right panel) adult skin double-stained for CD11b and Ly6G. In unirradiated skin, all CD11b+ cells were Ly6G– (left panel), but after UV, CD11b+Ly6G– and CD11b+Ly6G+ cells were detected (right panel). (D) Cytospins of FACS-sorted cells from UV-irradiated, adult animals. CD11b+Ly6G– cells (left panel) were a mixed population including lymphocytes, eosinophils, and large mononuclear cells, whereas CD11b+Ly6G+ cells (right panel) were neutrophils. (E) Time course, quantitated by FACS analysis, of infiltrating CD11b+ and Ly6G+ cells and loss of MHC Class II cells after UV. In adults, significant influx of CD11b+ and Ly6G+ cells was observed (P<0.001 compared with unirradiated control) and was maximal 48 h after UV, whereas loss of MHC Class II (P<0.01) reached a plateau by 24 h. In neonates, no significant changes in CD11b+, Ly6G+, or MHC Class II cells were detected. The data represent the mean of three to five independent experiments ± SEM.

 
Dose and wavelength dependence of UV-induced inflammatory infiltrate
Infiltration of Ly6G+ and CD11b+ cells into adult skin irradiated with the F40 sunlamp was UV-dose-dependent (Fig. 3A ). In strong contrast, no significant alterations to Ly6G+, CD11b+, or MHC Class II+ cells were detectable in neonatal wild-type or HGF/SF-transgenic skin with any UV dose from this lamp. These data further confirm the major difference in the inflammatory response in neonates and were observed when pups were irradiated at time-points between Days 3 and 7 of age (data not shown). Epidermal LC comprised 5.8% ± 1.08% of adult skin but only 1.3% ± 0.3% of neonatal skin, which may be a factor in the inability to detect a decrease of MHC Class II cells in UV-irradiated, neonatal skin.


Figure 3
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  		Figure 3. Dose and wavelength dependence of infiltration of CD11b+, Ly6G+, and a decrease of MHC Class II+ cells in adult skin. (A) FVB [solid symbols:wild-type (Wt)] and HGF/SF-transgenic [open symbols:transgenic (Tg)] mice were irradiated with UV doses as indicated from F40 sunlamps and skin analyzed by FACS 48 h later for CD11b, Ly6G, and MHC Class II, as for Figure 2E . In adults (circles), influx of CD11b+ and Ly6G+ cells was dose-dependent (P<0.001 compared with unirradiated control). No significant alterations to CD11b+, Ly6G+, or MHC Class II+ cells were detected in FVB or HGF/SF-transgenic neonates (triangles). The data represent the mean value of three to five independent experiments ± SEM. (B) Influx of CD11b+ and Ly6G+ and loss of MHC Class II+ cells in adult FVB mouse skin 48 h after irradiation with 6.4 kJ/m2 UVB (>96%, 280–320 nm) or 144 kJ/m2 UVA (>99%, 320–400 nm). No alterations were detectable with UVA, although UVB induced CD11b+ and Ly6G+ infiltrate and loss of MHC Class II cells (circled). (C) Dose response for influx of CD11b+ and Ly6G+ and loss of MHC Class II+ cells in adult (•) and neonates ({blacktriangleup}). UVB was effective in adults but not in neonates. Our previous studies had shown these doses of UVB initiated melanoma in HGF/SF transgenics, although UVA was ineffective.

 
As irradiation of HGF/SF-transgenic neonates with a specialized source of UVB (>96%, 280–320 nm) was significantly more effective at initiating melanoma than was the F40 sunlamp [12 ], we investigated whether irradiation with this specialized source could initiate an inflammatory infiltrate in neonates (Supplemental Fig. 3). Adult FVB mice irradiated with UVB showed a dose-dependent infiltration of Ly6G+ and CD11b+ cells with a concomitant decrease in MHC Class II+ cells (Fig. 3B and 3C) , which was also time-dependent (not shown). Although these UVB doses were highly effective at initiating melanoma in neonatal HGF/SF transgenics, they were completely ineffective at initiating an inflammatory infiltrate in neonates (Fig. 3B and 3C) .

As a control, adults and neonates were irradiated with UVA, which in contrast, was ineffective with no detectable changes in any of the three cell types, although the dose used (144 kJ/m2) was much higher. The UVA dose was chosen based on our previous studies [12 ] in which UVA was shown to be ineffective at initiating melanoma in neonatal, HGF/SF-transgenic mice, although the UVB doses we have used here did initiate melanoma. There was no detectable infiltrate of CD11b+ or Ly6G+ cells or detectable change in MHC Class II+ cells in adult skin in response to UVA (Fig. 3B) or in neonatal mouse skin (not shown).

Inflammatory responses to topical TNCB or to peritoneal TG are impaired in neonatal mice
Two additional inflammatory stimuli were investigated to establish if the deficiency in the inflammatory response was specific for UV radiation and/or for the skin. A significant influx of Ly6G+ neutrophils into the peritoneal cavity after injection of TG was observed in adult mice (Fig. 4A ), which was dose-dependent. Comparable with the findings in the skin, there was, however, no significant infiltrate in neonatal mice (Fig. 4A) . There was observed, however, a decrease at 4 h in the proportion of F4/80+ cells in adults and neonates, which was maintained at 24 h in adults but not in neonates.


Figure 4
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  		Figure 4. The sterile, peritoneal, inflammatory response and the inflammatory response to topical TNCB are absent in neonates, and topical application of TNCB is tolerogenic. (A) i.p. injection of TG induced an increase in the number of cells in the peritoneal cavity in adult (•) but not neonatal ({blacktriangleup}) FVB mice, 4–24 h after injection. The percentage of peritoneal Ly6G+ cells was increased significantly in adults (P=0.001) but not in neonates (P=0.1) with a sustained decrease in F4/80+ cells in adults (P=0.001) and a transient decrease (significant only at 4 h, P=0.02) in neonates. (B) Kinetics of infiltration of Ly6G+ and loss of MHC Class II+ cells in skin after TNCB treatment of FVB (solid symbols) and HGF/SF-transgenic (open symbols) mice. Adults (circles) showed significant Ly6G+ infiltration (P<0.001) and loss of MHC Class II+ cells (P<0.001), but these effects were absent in neonatal mice (triangles). The data in each panel represent the mean value of three to five independent experiments ± SEM. (C) CHS to TNCB determined as ear swelling ± SEM, 24 h after challenge in 9-week-old, FVB mice. Four to seven mice per group were not sensitized [negative control (CTRL)], sensitized only as adults (positive control), or first sensitized with TNCB as pups and resensitized as adults. Prior exposure to TNCB as neonates resulted in a significantly diminished, adult CHS response to TNCB (*, P=0.01, compared with positive control) but did not alter the adult CHS response to OX (P=0.6).

 
Neonatal and adult mice were treated topically with TNCB, a contact sensitizer, and FACS analysis of disaggregated skin carried out. Adult wild-type and adult HGF/SF-transgenic mice responded similarly to TNCB treatment with a neutrophil (Ly6G+) infiltration and decrease of MHC Class II+ cells (Fig. 4B) , 24–48 h after TNCB application. In contrast, in neonatal wild-type or neonatal HGF/SF transgenics, application of TNCB did not induce an inflammatory infiltrate (Fig. 4B) , confirmed by H&E staining (not shown). In response to TNCB, a cutaneous vascularization was observed in adult but not in neonatal skin (not shown).

Immune tolerance was initiated by neonatal exposure to TNCB
To investigate the functional significance of the lack of inflammatory infiltrate in neonates, 3- to 6-day-old neonates were contact-sensitized with TNCB on dorsal or ventral skin. Eight weeks later, animals were resensitized on ventral skin and the ensuing CHS determined. The CHS response to TNCB was decreased significantly in animals, which had prior neonatal TNCB exposure (Fig. 4C) , indicating a tolerogenic response was initiated neonatally. This decrease was similar in animals, which were neonatally sensitized initially on dorsal or on ventral skin (P=0.7). In contrast, neonatal sensitization with TNCB did not alter an adult response to a different sensitizer, OX (Fig. 4C) , indicating antigenic specificity of the tolerogenic response.

Chemotaxis to Gro-{alpha} was deficient, and expression of CXCR2 was decreased in neonatal Gr1+ cells
Differential counts of neonatal and adult peripheral blood (Table 1 ) indicated a higher proportion of neutrophils in neonates (P<0.01) with a significant population of band (immature) neutrophils (P<0.01) absent in adult blood. The proportion of lymphocytes in neonatal blood was significantly lower, but the proportion of monocytes was significantly higher (Table 1) . FACS analysis indicated a significant decrease of side- and forward-scatter parameters of neonatal compared with adult neutrophils, indicating a lower level of granularity and smaller size of neonatal granulocytes, which also had a decreased surface expression of Gr1+, confirming their immaturity.


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  		Table 1. Neonates Have a Higher Proportion of Blood Neutrophils Than Adults

 
Chemotaxis to the CXC chemokine Gro-{alpha} (CXCL1, KC, N51)—also a potent melanoma growth factor—was not detectable in neonatal Gr1+ cells, although adult cells showed significant migration in response to the chemokine (Fig. 5A ). It is surprising that chemotaxis to the bacterial product fMLP was enhanced significantly in neonatal Gr1+ cells in comparison with adults (Fig. 5B) , indicating that neonatal granulocytes were capable of chemotaxis and that there was a selectivity to the chemotactic defect. There were no detectable differences in migration to either agent between adult wild-type and HGF/SF-transgenic animals. The surface expression of the receptor for Gro-{alpha}, CXCR2, in neonatal Gr1+ cells was lower than in adults with a MFI threefold lower than that of adult cells, and CXCR2 expression was heterogeneous (Fig. 5C) , although CD11b expression was similar to adults (Fig. 5D) . The decreased expression of CXCR2 in neonatal cells is consistent with the chemotactic deficiency to Gro-{alpha} observed in these cells.


Figure 5
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  		Figure 5. Impaired chemotaxis to Gro-{alpha} and decreased expression of CXCR2 in neonates. (A and B) Chemotaxis of blood Gr-1+ neutrophils to Gro-{alpha} (10 ng/ml) or fMLP (10–6M) in neonatal (nn) FVB mice and adult HGF/SF transgenics expressed as percentage of chemotaxis in adult FVB mice. *, Compared with adults, neonatal cells migrated significantly less to Gro-{alpha} but significantly better to fMLP (P<0.001). (C and D) Expression of CXCR2 on Gr1+ cells from neonates (gray line) was heterogeneous and expressed at lower levels than in adults (black line); negative control, dotted line. Mean fluorescence intensity (MFI) was threefold lower for neonatal compared with adult cells (MFI values: neonatal, 64; adult, 195). In contrast, CD11b levels were similar in adult and neonatal cells.

 
DISCUSSION

We have found that neonatal mice have a profound defect in the inflammatory response, which persists for at least 7 days after birth. This defect was observed in wild-type FVB mice and melanoma-prone, FVB-HGF/SF-transgenic animals. The lack of an inflammatory infiltrate in neonatal skin in response to stimuli which are highly inflammatory in adults, is, we propose, a major contributor, not only to neonatal-immune tolerance but also to the successful outgrowth of neonatally initiated cancers. This is the first report of a defect in neonatal mouse skin in response to environmental inflammatory agents, notably UV radiation, a well-established environmental carcinogen. Our observation that this defect occurs also in response to TG in the neonatal peritoneal cavity further emphasizes its significance in neonatal biology.

Although previous investigations have not carried out detailed, quantitative dose-responses and kinetics as we have described, our data in adult mouse skin are in general agreement with earlier studies. In humans (reviewed in refs. [15 , 26 ]) and in mice [27 , 28 ], neutrophils are a major component of UV-induced inflammation. Infiltration of dermal CD11b+ inflammatory macrophages, precursors of dermal dendritic APC [28 , 29 ], has also been reported in human and mouse skin in response to UV. We observed a small increase in a mixed population of CD11b+Ly6G– cells after UV (Fig. 2) , which may have included this macrophage subset. The major inflammatory component was, however, neutrophilic (CD11b+Ly6G+). We also observed a loss of MHC Class II cells in adult skin, likely predominantly epidermal LC, in response to UV or to TNCB, effects well described previously [24 , 30 , 31 ]. We did not, however, observe any significant depletion of MHC Class II cells in neonatal skin in response to UV or to TNCB, further emphasizing the differences between adult and neonatal responses. We did not observe any changes in skin cell populations in response to UVA, in broad agreement with previous studies, although one investigation in humans [32 ] with UVA doses approximately fourfold higher than those we used did report loss of epidermal LC, albeit without inflammatory infiltrate. Studies using isolated UVB have been little reported, but our findings—that UVB alone, without the contaminating wavelengths present in conventional broadband lamps, initiated an inflammatory infiltrate—were in agreement with in vivo data in humans [33 ] and in mice [34 ], for which erythema was the endpoint.

A decrease in the number of circulating neutrophils in neonates was not responsible for the deficiency in inflammatory infiltrate, as neonatal peripheral blood contained more than twice the percentage of neutrophils in adult blood. Rather, neonatal Gr1+ blood cells, in contrast to those from adults, were deficient in chemotactic responsiveness in vitro to the chemokine Gro-{alpha} and exhibited a decreased expression of the receptor for Gro-{alpha}, CXCR2. Gro-{alpha} is the major chemoattractant in vivo in response to the inflammatory stimuli we have used and is also responsible for neutrophil arrest [35 ]. Gro-{alpha} is the major neutrophil attractant in UV-irradiated skin [36 ] and in skin in response to TNCB application in naïve mice [37 ]. In TG-induced peritonitis, inhibition of CXCR2 impaired neutrophil arrest [38 ], implicating Gro-{alpha} also in this form of inflammation. Recruitment of neutrophils to the skin in response to the tumor promoter 2-O-tetradecanoylphorbol-13-acetate is completely dependent on CXCR2 [39 ]. Further, CXCR2 is the receptor, not only for several CXC ligands of the Gro/melanoma growth-stimulatory and IL-8 family but also for CXCL5, -6, and -7, which function in attraction, chemotaxis, and activation of neutrophils [40 ], indicating its central role in neutrophil responsiveness. Thus, the decreased expression of the CXCR2 receptor was a major factor in the profound lack of the inflammatory response observed.

It is interesting that as neonatal neutrophils were able to migrate successfully in vitro to the bacterial peptide fMLP and had normal cell surface expression of CD11b, these neutrophils were competent to respond to some signals, indicating major aspects of the chemotactic signaling pathways were intact. It is well established in humans that neutrophils from newborn infants have deficient chemotaxis [41 42 43 ]. A decreased chemotactic response of neonatal human neutrophils to a wide range of chemokines was reported [42 ]. Blood chemokine levels, however, were reported to be similar in neonates and adults [43 ]. There is, however, no consensus on the mechanisms responsible for the neonatal chemotactic deficiency in infants, which has been attributed to a variety of factors including altered expression of selectins and/or integrins, to abnormal receptor distribution in response to the chemotactic stimulus, to altered receptor shedding, and to abnormalities in the neutrophil cytoskeleton [41 , 44 ].

Neonatal exposure to antigen resulted in antigen-specific immune tolerance in our studies. Neonatal mice topically sensitized with TNCB were resistant to subsequent resensitization and challenge with TNCB, but responses to another antigen, OX, were unaffected. These observations are in agreement with published studies [45 ]. Neonatal epidermal LC in mouse skin have been reported to express lower levels of MHC Class II antigen and DEC 205, necessary for effective antigen presentation, and LC from the draining lymph nodes of contact-sensitized, neonatal mice expressed lowered levels of costimulatory molecules [45 , 46 ] Although neonatal dendritic cells (DC) have been reported as constitutively producing lower levels of critical cytokines, notably IL-12 [47 , 48 ], impairing their ability to stimulate Th1 responses, stimulation of neonatal DC with appropriate maturation signals has enabled effective antigen presentation [49 ]. We propose that the lack of inflammatory infiltrate we have demonstrated here in skin in response to UV radiation or to application of the contact sensitizer TNCB is a major factor in the inability of neonatal skin to provide DC maturational signals. Inflammatory neutrophils are well described as playing a major role in delivering maturational signals to DC by direct cell surface interactions through DC-SIGN and by the release of inflammatory cytokines, notably TNF [50 , 51 ]. Further, DC from neutrophil-depleted mice are deficient in cytokine production in response to an in vivo infection [50 ]. The selectivity of the chemotactic defect in neonatal neutrophils that we have described, with a deficient chemokine response but an intact response to the bacterial product fMLP, is consistent with a model of neonatal immune tolerance [4 , 48 ] in which inflammatory responses to environmental antigens are blunted because of the potential collateral, inflammatory damage to developing organs, but essential responses to life-threatening, infectious agents are necessarily retained.

In neonatal skin, although there was no infiltrate of inflammatory cells, some UV responses were intact. Comparable levels of UV-induced DNA damage were observed in adults and neonates. It is notable that edema was readily detected in neonates, as in adults, indicative of blood-vessel leakage. Erythema and increased cutaneous vascularization, resulting from vasodilation and angiogenesis, although observed in adult skin, were not detectable in neonatal skin. This was surprising, as vascular endothelial growth factor (VEGF), considered the major peptide responsible for vascular leakage, also has well-described, angiogenic and vasodilatory properties [52 ]. VEGF has been demonstrated to be released from mouse keratinocytes and from mouse and human skin [16 , 53 ] in response to UV, in particular, in response to UVB rather than UVA [54 ] and has been shown responsible for edema, angiogenesis, and vasodilation in UV-irradiated adult mouse skin [55 ], although there are no data in neonatal skin. It has been a subject of debate whether vascular permeability and angiogenesis can be separated [52 ]. Our data indicate that these events do not necessarily occur together, in agreement with findings that specific Src kinase requirements differ for vascular permeability and angiogenesis [56 ], and soluble neuropilin, a VEGF inhibitor, inhibits vascular permeability but not angiogenesis [57 ]. Angiogenesis is essential for development, including postnatal development and VEGF inhibition although it has little effect in adults, in neonates results in death [58 ]. The absence of detectable changes in cutaneous vascularization—as distinct from permeability—in neonates may reflect a tighter control on vasodilation and angiogenic processes. The lack of an inflammatory infiltrate in neonates may further prevent an amplification of the vascular response by vasodilatory and angiogenic factors released from infiltrating cells.

In conclusion, we have identified a profound defect in the inflammatory response in neonatal mice, notably in response to the environmental carcinogen UV radiation. This finding has significance for mechanisms of neonatal-immune tolerance and may be relevant to neonatal susceptibility to carcinogenesis.

ACKNOWLEDGEMENTS

We thank Dr. Glen Merlino for the HGF/SF-transgenic mice, Dr. David Leitenberg for manuscript review, Christopher Shumate, Jesse Bahn, Ifeanyi Okwumabua, and Russell Morton for technical assistance, and Tomasz Glubisz for help with graphic design. These studies were funded by NIH CA53765 and 92258 (F. N.) and AI067254 (S. C.).

FOOTNOTES

1 Current address: Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Biophysics, ul. Gronostajowa 7, 30-387 Krakow, Poland. Back

Received December 13, 2006; revised January 19, 2007; accepted February 20, 2007.

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