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First published online 26 July 2005
doi: 10.1242/jcs.02474

Journal of Cell Science 118, 3685-3694 (2005)
Published by The Company of Biologists 2005
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Accelerated dendritic-cell migration and T-cell priming in SPARC-deficient mice

Sabina Sangaletti1, Lucia Gioiosa1, Cristiana Guiducci1, Gianluca Rotta2, Maria Rescigno2, Antonella Stoppacciaro3, Claudia Chiodoni1 and Mario P. Colombo1,*

1 Immunotherapy and Gene Therapy Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milano, Italy
2 Department of Experimental Oncology, European Institute of Oncology, 20141 Milano, Italy
3 Department of Experimental Medicine and Pathology, 2nd Faculty of Medicine, University of Rome "La Sapienza", Roma, Italy



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  		Fig. 1. Enhanced CHS in SPARC-knockout mice. SPARC+/+ and SPARC–/– mice were sensitized with DNFB on the dorsal side of the right ear on day 0. Five days later, mice were challenged on the left ear. Control mice from the two strains were treated only with the challenge dose of DNFB. Panel A shows ear swelling measurements (mean±s.d.) at 24 and 48 hours after challenge for each group of mice. Mean values were significantly different at both time points (P<0.001). Results are representative of five independent experiments. Panel B shows the inflammatory response in sections of the challenged ear of SPARC+/+ and SPARC–/– mice. Tissues were harvested 24 hours after DNFB challenge, fixed in 10% neutral buffered formalin and sections stained with hematoxylin and eosin. Owing to extensive swelling, only a portion of the SPARC–/– section is shown (cartilage is below the cut line of the figure and was omitted to ensure a magnification matching that of the control panel). Inflammatory infiltrate appears to be more abundant in the SPARC–/– section.

 


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  		Fig. 2. The same numbers of Langerhans cells (Langerin/CD207+) but different collagen content characterize the ear skin of SPARC+/+ and SPARC–/– mice. (A) The distribution of Langerin/CD207+ cells in the epidermal sheet from untreated SPARC+/+ and SPARC–/– mice is very similar. Cell counting and morphometry revealed essentially the same numbers of LCs in the epidermal sheet of the two strains (SPARC+/+=50.9±7.5 and SPARC–/–=53.8±4.4 in 10 randomly chosen fields, n=5). (B) Total ear collagen content and distribution are shown in cross-sections of the middle auricles by trichromic stain. SPARC+/+ ear pinna is characterized by a greater amount of collagen on both sides of the cartilage; increased thickness of the internal face of auricles and minor evidence of external auricle subcutaneous fat and muscle structures are seen compared with SPARC–/– ear pinnae.

 


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  		Fig. 3. Distribution of Langerhans/CD207+ cells into the draining LN of SPARC+/+ and SPARC–/– mice after DNFB sensitization. Draining LNs were removed 24 and 48 hours after sensitization, sectioned and stained with mAb to Langerin/CD207. Langerin/CD207+ cells were counted under a microscope with a 20x objective. Panel A shows representative Langerin staining of draining LN from SPARC+/+ and SPARC–/– mice collected 24 and 48 hours after DNFB painting. Panel B shows the number (mean±s.d.) of Langerin/CD207+ cells counted in 10 randomly chosen fields from six independent preparations for each SPARC+/+ and SPARC–/– experimental group.

 


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  		Fig. 4. Migration of LX-bearing cells in the draining LN and consequent increase in LN cellularity. (A) SPARC+/+ and SPARC–/– were injected with 107 green-LX-microspheres in the dorsal side of the right ear, which was painted with DNFB 30 minutes later. Draining LNs were collected after 8 and 24 hours and cells were immunolabeled with PE-conjugated anti-I-Ad/I-Ed for two-color FACS analysis. A higher number of green-LX-loaded/I-Ad/I-Ed+ cells were found in draining LNs from SPARC–/– than from SPARC+/+ mice at both 8 and 24 hours after treatment. (B) Morphology of green-LX-loaded/I-Ad/I-Ed+ cells in the draining LN reveals the typical hairy and veiled appearance of DCs. (C) Total cell number in the draining LN of treated mice. Migration of green-LX-loaded/I-Ad/I-Ed+ cells in the draining LN is associated with increased LN cellularity which is more extensive in SPARC–/– mice.

 


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  		Fig. 5. Migration of BM DCs does not depend on their SPARC genotype. We pre-loaded in vitro BMDCs from SPARC+/+ and SPARC–/– mice with green- or red-LX microspheres, respectively (or vice versa), before co-injecting them (5x105 each) intradermally into the ear dorsal side of SPARC+/+ or SPARC–/– mice. Draining LN were collected after 48 hours and analyzed by FACS. (A) An example of SPARC+/+ green-LX- and SPARC–/– red-LX-loaded DCs in draining LN from SPARC+/+ mice. (B) Cumulative data of all tested combinations. A higher number of green- or red-LX-loaded DCs was found in draining LNs of SPARC–/– mice, regardless of the SPARC genotype of the LX-loaded DCs. Each bar represents the mean value of 14 LNs analyzed (7 mice/group).

 


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  		Fig. 6. Effect of DC migration on T-cell priming. Panel A shows the accelerated CHS occurring in SPARC–/– mice. DNFB-sensitized SPARC+/+ and SPARC–/– mice were challenged with DNFB starting 48 hours after sensitization and every other day until day 5. Ear swelling was measured 24 hours after challenge. At every time point, ear swelling was significantly higher (P<0.05) in SPARC–/– than in SPARC+/+ mice. Panel B shows DTH elicited in SPARC–/– but not SPARC+/+ challenged mice 48 hours after OVA sensitization. Panel C shows the effect of DC migration on priming of OVA-specific transgenic T cells. CFSE-labeled KJ+CD4+CD25– cells were adoptively transferred into SPARC+/+ and SPARC–/– mice. Mice were immunized 8 hours later by injection of OVA protein into the ear pinna. Draining LNs were isolated at the indicated time points after immunization and stained with KJ-126-PE Ab. the histogram show the analysis from a representative mouse at each time point for SPARC+/+ and SPARC–/– groups. See Table 1 for quantitative data analysis. M1 and M2 indicate non-divided and divided cells, respectively.

 


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  		Fig. 7. Migration of Langerhans cells from skin explants. Ear skins (dorsal halves, cartilage-free) obtained from SPARC+/+ and SPARC–/– mice were cultured in 24-well tissue culture plates (one ear half per well) in the presence of rGM-CSF. (A) Cells that emigrated into the culture medium, were characterized by FACS with monoclonal antibody to Langerin/CD207 and DEC-205 as shown for SPARC+/+ explants as an example. (B) The mean number of Langerhans cells obtained from the ear skin of SPARC–/– mice, was significantly higher than that obtained from SPARC+/+ mice both at 24 and 48 hours of culture. The mean number of Langerhans cells was calculated as total number of collected cells multiplied by the fraction of Langerin/CD207+ stained cells. Results are representative of three independent experiments.

 


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  		Fig. 8. CHS in chimeric mice depends on environmental SPARC. (A) SPARC+/+>SPARC–/– chimeric mice expressing SPARC only in their BM compartment and reciprocal SPARC–/–>SPARC+/+ chimeras were obtained by using, respectively, (CB6)F1 mice as donor or recipient in BM transplantation experiments. To ensure that LCs were of donor origin, chimeras were UV-irradiated 8 weeks later to allow LC renewal from BM precursors. Three weeks after radiation, chimeric and control mice (UV-irradiated or not) were tested for CHS. (B) Immunostaining of MHC-II haplotype in the epidermal sheet of SPARC+/+>SPARC–/– chimera before and after UV-irradiation at the indicated time points. (C) The degree of CHS was dependent on the host rather than donor SPARC genotype, with greater CHS in SPARC+/+>SPARC–/– chimeras, which did not differ from SPARC–/– control mice. No differences in ear swelling degree were detected in UV-irradiated and non-irradiated mice after replenishment of LCs from BM precursors, further confirming the role of environmental SPARC.

 

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© The Company of Biologists Ltd 2005