Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Splenic and immune alterations of the Sparc-null mouse accompany a lack of immune response

Genes & Immunity volume 8, pages 262–274 (2007)Cite this article

Abstract

Sparc-null mice have been used as models to assess tumor-host immune cell interactions. However, it is not known if they have a competent immune system. In this study, the immune systems of Sparc wild-type and null mice were compared. Mice were assessed for differences in total body weight, spleen weight and spleen-to-body weight ratios. Spleens were compared with respect to morphology, and Sparc, Ki-67, MOMA-1 and IgM expression. Immune cells in blood, bone marrow and spleen were assessed by blood smears, automated blood panel, and flow cytometry. Additionally, the ability of Sparc-null mice to respond to immune challenge was evaluated using a footpad model. The morphological and immunohistochemical results indicated that Sparc-null spleens had more white pulp, hyperproliferative B cells in the germinal centers, and decreased marginal zones. Sparc-null spleens lacked normal Sparc expression in red and white pulp, marginal zones, endothelial and sinusoidal cells. By flow analysis, B cells were decreased and T cells were increased in the bone marrow. Finally, Sparc-null mice were unable to mount an immune response following footpad lipopolysaccharide challenge. These data confirm that Sparc-null mice have an impaired immune system.

Download PDF

Introduction

SPARC (osteonectin, BM-40) is a secreted matricellular protein involved in the modulation of cell adhesion and motility. Sparc-null mice have been generated1, 2 to examine its roles in development and disease. The first major phenotype reported was the development of cataracts 6–8 months postnatally.1, 2 Since then, additional phenotypes have been described including osteopenia,3 increased fat deposition,4 altered wound healing5 and altered collagen deposition.6

SPARC has also been implicated in tumor progression. However, seemingly disparate results have been obtained, depending on the tumor type studied.7 For some cancers, such as ovarian cancer, the loss of SPARC is associated with progression8, 9 and the re-expression of SPARC in ovarian tumor cells induces apoptosis.10 Similarly, aggressive neuroblastomas have little SPARC expression in the tumor cells, whereas SPARC is expressed in the surrounding stromal cells. Treatment of the tumors with SPARC peptides in vivo inhibited tumor growth.11, 12 In contrast, SPARC is highly expressed in melanoma13 and gliomas.14 The inhibition of SPARC expression in melanoma cells15 and the overexpression of SPARC in glioma cells16, 17 promoted decreased adhesion and increased migration, respectively, suggesting that increased SPARC expression in these cancer types promotes invasion. That SPARC may be associated with good prognosis for some cancers, but worse prognosis for others, may in fact be due to the difference in expression patterns, that is, tumor cells versus stromal cells, the latter including resident immune cells. The generation of Sparc-null mice has provided a means to examine the role of Sparc in these specific compartments.

In a study by Sangaletti et al.,18 SPARC-expressing mammary tumor cells were injected into the mammary fat pad of Sparc-null and wild-type (WT) mice. Tumor cells injected into WT mice developed tumors with lobular structures having well-defined septa expressing high levels of collagen IV, and infiltrating immune cells. Sparc was expressed in both immune and tumor cells. When tumor cells were injected into Sparc-null mice, where Sparc expression was limited to the tumor cells, the tumors displayed an increased number of infiltrating immune cells. However, the resultant tumors were less well structured, having smaller septa and less collagen IV. These data indicated that Sparc produced by the host leukocytes determined the assembly and function of tumor-associated stroma through the organization of collagen type IV. In contrast, several other studies indicate that a loss of host Sparc augments tumor progression,9, 19, 20 suggesting fundamental differences in the role of Sparc and the regulation of growth in different tumor types. However, all of the studies reported a decreased extracellular matrix (ECM) and an increase in infiltrating macrophages with the loss of host Sparc. These studies however, did not address the role of Sparc in the leukocytes with respect to their ability to modulate an immune response.

That Sparc expression by tumor cells helps the cells escape from immune surveillance is supported by a study by Alvarez et al.21 They demonstrated that the inhibition of Sparc expression in melanoma cells implanted into nude mice promoted polymorphonuclear (PMN) leukocyte recruitment to the tumor, and importantly, that the PMNs suppressed tumor growth. However, PMNs are the first line of immune defense, and the study is limited by the inability to study a fully competent immune system using a nude mouse model.

Several studies suggest possible differences in underlying immune responses between Sparc-null and WT mice. Increased neutrophil and leukocyte infiltration was observed in bleomycin-induced peritonitis22 and in TPA-induced skin inflammation18 in the knockouts. However, these studies did not address the underlying mechanism with regards to the need for Sparc expression by the leukocytes or in the ECM in the differences observed. In a follow-up study, Sangaletti et al.23 examined Langerhan cell (LC) migration and the ability to impact T-cell priming in cutaneous contact hypersensitivity (CHS) in Sparc WT and null BALB/cAnNCrl mice, and in WT and null mice previously irradiated and rescued with null or WT bone marrow cells, respectively. These studies demonstrated that the loss of Sparc expression did not alter the inherent ability of the LCs to migrate. However, the lack of Sparc expression altered the ECM, thereby decreasing the physical barrier confronting the LCs and hastening migration to lymph nodes, which accelerated the onset of T-cell priming. In this case, loss of Sparc in the stroma rather than loss of expression by immune cells appeared to be important in controlling the extent of CHS. In contrast, Sparc-expressing SKH-1 mice demonstrated greater CHS response to oxazolone than SKH-1/Sparc-null mice.24 Differences in outcome may be due to the use of different strains of mice and/or different sensitizing agents.

Therefore, although these studies have examined the role of Sparc expression in local immune reactions, either by immune cells or by tumor cells, none have addressed the global significance of Sparc in the development of a competent immune system.

We have also been breeding Sparc-null and WT mice. At two institutions, we observed that several of the aged Sparc-null mice that were injured developed infections that did not heal, requiring the mice to be killed. These observations suggested differences in the ability of the immune systems to respond to infection, suggesting that Sparc-null mice were immune compromised. This could potentially impact the interpretation of tumor-immune cell interactions. This prompted us to analyze the immune system in Sparc WT and null mice that had not been in any way treated or subjected to tumor implant. We examined the mice for differences in total body weight, and the weights, morphology, and Sparc, Ki-67, MOMA-1 and IgM expression in the spleens. Furthermore, differences in immune cell populations were assessed by blood smears, automated blood panel, and flow cytometry of bone marrow, blood and spleen compartments. Finally, response to immune challenge was assessed.

Results

Generation of Sparc WT and null mice

Polymerase chain reaction (PCR) genotyping for the Sparc-null and WT alleles demonstrated the generation of both populations (Figure 1a). The loss of Sparc expression in null spleens is represented by Western blot analysis using anti-Sparc antibody (Figure 1b).

Figure 1
figure 1

Sparc-null (Null) mice have a larger spleen and a greater spleen-to-body weight ratio compared with Sparc WT (WT) mice. (a) PCR genotyping of null and WT mice using Sparc-specific primers as described in Materials and methods. ^ – The lanes are on the same gel, but moved closer. (b) Western blot analysis demonstrating the lack of Sparc protein in the spleen from null versus WT mice. ^ – The ladder is on the same gel, but moved closer. Actin was used as a loading control. (c and d) A comparison of total body weight indicates no differences between null and WT genotypes (c) or between genders/genotype (d). (e and f) A comparison of wet and dry weights for thymus organs indicates no differences between null and WT genotypes (e) or between genders/genotype (f). A comparison of spleen wet and dry weights indicates that the spleens are larger in null compared with WT mice (g, inset illustrating the spleens immediately after removal). Differences in wet and dry weights are maintained for both genders within a genotype (h). The ratio of spleen weight to 100 g total body weight is greater for null mice than for WT mice (i). This difference in ratio is maintained between genders (j), n=17 null and n=11 WT; of these, n=14 male subjects, n=14 female subjects.

Full size image

Sparc-nulls have larger spleens and a greater spleen/body weight ratio

We saw no statistically significant difference in the overall body weight between Sparc WT and null mice when assessed by genotype (Figure 1c), or by gender within a genotype (Figure 1d). No differences in wet or dry weights were observed for any organs from WT and null mice, including another immune organ, the thymus (Figure 1e and f), except for the spleens (Figure 1g and h). Gross changes in size are illustrated in the Figure 1g inset. The null mice had higher spleen wet weights (approximately 1.5-fold (Figure 1g)), consistent for both genders (Figure 1h). A similar trend was observed for dry weight. In addition, the larger spleens represented a greater percentage of the total body weight (Figure 1i), also consistent for both genders (Figure 1j).

Localization of Sparc expression in WT spleens

Assessment of Sparc expression by Western blot analysis suggested that the protein might be expressed in the spleens. Alternatively, it could be a signal derived from trapped circulating cells, such as platelets. Immunohistochemical analysis indicated that Sparc is normally expressed sparsely in endothelial cells within the lymphoid follicles and marginal ones, and more profusely in sinusoids and cells of the red pulp (Figure 2e–h). Loss of Sparc expression was confirmed in the null spleens (Figure 2a–d).

Figure 2
figure 2

Sparc localization in WT spleens and confirmation of loss in Sparc-null spleens. (a–h) Representative formalin-fixed, paraffin-embedded spleen sections from Sparc-null (a–d) and Sparc WT (e–h) mice were immunohistochemically stained for Sparc or CD31 (d, h; top panels) expression. Magnifications are × 40 (a, e), × 100 (b, f) and × 400 (c, d, g, h). The regions of red pulp (rp), white pulp (wp) and marginal zones (mz, arrows) indicated in a and e, are magnified in (b–c) and (f–g), respectively. Sparc expression is observed in endothelial cells within the white pulp (f, g; inset) and in the sinusoidal cells (f, arrowheads; g, arrow indicating magnified region in f). A punctate staining pattern is observed throughout the red pulp (g) and white pulp (g inset). Sparc expression is absent in the Sparc-null spleens as expected (a–d). Rarely, an immunoreactive cell is observed suggesting minimal non-specific cross-reactivity (c, arrow; WP: inset). A background signal is observed in both Sparc-positive and Sparc-negative spleens due to hemosiderin granules. Endothelial cells were identified using CD31 antibody (d, h: top panels). Endothelial cells in WT spleens (arrows) are Sparc-positive (h; lower panel; inset: magnification of an endothelial cell). Endothelial cells of Sparc-null spleens are negative (d: lower panel) (n=4/genotype).

Full size image

Sparc-null spleens differ in morphology

As cells within the spleen normally express Sparc, we compared WT and null spleens to determine whether morphological changes accompanied the loss of its expression. In the Sparc-null spleens, there was expansion of B-cell-rich nodular white pulp and an increased number of secondary lymphoid follicles (Figure 3a–c) in comparison to the WT spleens (Figure 3d–f). The follicular lymphoid hyperplasia in the null spleens was characterized by prominent germinal centers with numerous centroblasts, centrocytes, mitotic figures, and readily apparent tingible-body macrophages. In addition, the marginal zones appeared distinctly attenuated in the null spleens. The T-cell-rich periarteriolar lymphatic sheaths (although perhaps slightly hypercellular) were unremarkable. No differences in morphology were observed for any other organs.

Figure 3
figure 3

Spleens from Sparc-null mice are morphologically different. (a–f) H&E-stained, formalin-fixed, paraffin-embedded and sectioned spleens from Sparc-null (a, b, c) and Sparc WT (d, e, f) mice. In the null spleens, the secondary (hyperplastic) follicles are better circumscribed than those in the WT spleens, with germinal centers containing numerous centroblasts (large nuclei, prominent nucleoli, and appreciable, pink cytoplasm) and centrocytes. There are frequent tingible-body macrophages (arrows) and mitotic figures (arrowheads). For the WT spleens, the primary lymphoid follicles are rather ill-defined and sparsely cellular, with prominent marginal zones. Both tingible-body macrophages and mitotic figures are inconspicuous. There were no gender differences within a genotype for male subject (a, c and d, f) or female subject (b, e) spleens. Images are at × 600 magnification (a, b, d, e). Regions in a and d were further magnified (× 1200) in (c, f) respectively, using Photoshop software), n=8/genotype.

Full size image

Sparc-null spleens have greater numbers of proliferative follicular B cells

The follicular hyperplasia observed by morphology was confirmed and quantitated using the proliferation marker Ki-67. The Sparc-null spleens demonstrated significantly greater numbers (approximately 3.6-fold) of proliferating germinal center cells (Figure 4a) in comparison with the WT (Figure 4c). On average, the WT spleens had an average of 166.5±34 SE Ki-67+ cells/follicle in comparison with the null spleens that had an average of 602.7±22 SE Ki-67+ cells/follicle (P<0.0001; Figure 4e). Analysis of Ki-67 staining within the red pulp indicated no statistical differences between WT and null spleens (data not shown).

Figure 4
figure 4

Sparc-null mice have spleens with greater numbers of proliferating germinal center B cells, more white pulp and smaller marginal zones. (a and c) Ki-67 expression in formalin-fixed, paraffin-embedded and sectioned spleens from Sparc-null (a) and Sparc WT (c) mice. Greater numbers of proliferative B cells were observed within the germinal centers of Sparc-null spleens. Images are at × 40 magnification, n=4/genotype. (b and d) H&E-stained formalin-fixed, paraffin-embedded sections of spleens from Sparc-null (b) and Sparc WT (d) mice. Images are at × 100 magnification of regions identified by bars in (a and c). Bars indicate marginal zones, which are attenuated in the Sparc-null spleens, n=4/genotype. (e) The number of Ki-67-positive germinal center B cells/follicle was counted and averaged/spleen. The averages/genotype were then plotted, n=4/genotype. (f) The percent area of white pulp/spleen was measured. The averages/genotype were plotted, n=4/genotype. (g) The number of cell layers/marginal zone/follicle was counted and averaged/spleen, n=4/genotype.

Full size image

Sparc-null spleens have more white pulp per given area

To determine whether the loss of Sparc altered the overall amount of white pulp, the percentage of splenic tissue comprised of white pulp was measured. On average, the Sparc-null spleens had more white pulp than normal spleens (P=0.0245; Figure 4f).

Sparc-null spleens have smaller marginal zones

The splenic marginal zones were assessed by counting the cell layers within this compartment. The bars indicate marginal zones in Ki-67-stained (Figure 4a and c) and adjacent hematoxylin and eosin (H&E)-stained (Figure 4b and d) sections. WT spleens had larger marginal zones (Figure 4d) with an average of 12.2 cell layers (Figure 4g) in contrast to the Sparc-null marginal zones (Figure 4b), which had an average of 7.4 cell layers (Figure 4g). These results are significantly different (P=0.0041).

Immunohistochemical assessment of marginal zones development

To further evaluate marginal zone formation, spleens were immunohistochemically stained with MOMA-1 and anti-IgM antibodies. MOMA-1 antibody was used to delineate marginal metallophilic macrophages (MMM) that line the inner border of the sinus margin and, in effect, demarcate the boundary between white pulp and marginal zone. Anti-IgM antibody was employed to identify marginal zone B cells, which tend to express high levels of IgM. In tandem, these immunostains demonstrated a normal complement of MMM in both WT and null spleens, and confirmed the decrement in IgM+ marginal zone B cells just outside the MMM in the Sparc-null spleens (Figure 5).

Figure 5
figure 5

Sparc-null spleens have enlarged white pulp and fewer IgM-expressing marginal zone B cells. (a–h) Representative Sparc-null (a–d) and WT (e–h) spleens were immunohistochemically stained with MOMA-1 antibody to detect MMM (red) and with anti-IgM to detect marginal zone B cells (blue). Note: IgM expression is not restricted to marginal zone B cells. Magnifications are × 40 (a, e), × 100 (b, f), × 200 (c, d) and × 400 (d, h). (b–d and f–h) Images are increasing magnifications of the red pulp (rp), white pulp (wp) and marginal zones (mz, arrrows) indicated in (a and e), respectively. Note the increased size of follicles (a versus e) and the decrease in IgM-positive marginal zone B cells (a–d versus e–h) in the Sparc-null versus WT spleens, respectively, n=3/genotype.

Full size image

Quantitation of white blood cells in blood smears

Blood smear analysis permits not only a count of the numbers of cells, but also allows an assessment of blood cell morphology. There were no significant differences in the blood cell morphology between Sparc-null and WT mice. In addition, there were no significant differences in total counts for monocytes, granulocytes or lymphocytes as assessed by genotype (Figure 6a) or when split by gender (Figure 6b). Although the slightly higher number of lymphocytes in the Sparc-null mice was not significant, it did suggest the possibility of an increase in a sub-population of lymphocytes.

Figure 6
figure 6

Blood smears demonstrate a trend towards an increased lymphocyte population in Sparc-null mice. (a) Blood smears were counted and the percentage of granulocytes, lymphocytes and monocytes are illustrated for Sparc-null (Null) versus Sparc WT (WT) mice. (b) The cell percentages were plotted by gender. Although no statistically significant differences are observed, the data suggest a trend for decreased granulocytes and increased lymphocytes in the Sparc-null blood smears, n=17 null, n=11 WT.

Full size image

Hematologic analyses

An automated blood panel was performed to define the cellular components more clearly (Table 1). In agreement with the blood smears, no differences were observed for monocytes, and neutrophils were significantly lower in the Sparc-null mice. In addition, no differences were observed between eosinophils and basophils. In agreement with the trends observed by the blood smears, this analysis demonstrated a slight but significant increase in lymphocytes of the Sparc-null mice, even though the total white blood cells (WBCs) were significantly lower (Table 1). Owing to the gross morphological differences in the spleen, subsequent analyses focused on the lymphocyte compartment.

Table 1 Hematologic assessment
Full size table

Flow cytometric immunoanalysis of Sparc-null and WT lymphocytes isolated from bone marrow, blood and spleens

We performed flow cytometric immunoanalysis on bone marrow, blood and spleens to determine if there were differences in major lymphocyte subsets in the Sparc-null versus WT mice. Statistically significant differences were observed only for bone marrow lymphocytes, which revealed a relative increase in CD3+ cells (T cells) and a relative decrease in CD19+ cells (B cells) in the Sparc-null mice (Table 2). A representative flow cytometric analysis is illustrated for CD3 and CD19 markers (Figure 7).

Table 2 Bone marrow, blood and spleen lymphocyte subset marker analyses
Full size table
Figure 7
figure 7

Characterization of Sparc-null lymphocyte sub-populations in bone marrow relative to sub-populations found in Sparc WT bone marrow. (a, b) Representative FACS analysis of total bone marrow mononuclear cell (BMMC) populations from (a) Sparc-null and Sparc WT mice (b). The total BMMC population was gated on the lymphocyte population, and the total CD3+ and CD19+ populations were determined. Lymphocytes isolated from Sparc-null mice have increased CD3+ populations and decreased CD19+ populations relative to the Sparc WT mice. Note: M1 region is based on negative control staining with fluorochrome-labeled secondary antibodies in the absence of primary antibodies, n=10/genotype.

Full size image

Ability to activate an immune response

As the data suggested alterations in immune cell numbers in the different compartments, the ability of the immune system of Sparc-null mice to activate a response was evaluated by the footpad swelling response. As can be seen in Figure 8, WT mice had a normal swelling response to the injection of lipopolysaccharide (LPS) over that of phosphate-buffered saline (PBS), with significant differences observed at 72 and 96 h. However, no response was observed to either LPS or PBS by the Sparc-null mice in comparison with the WT mice, indicating the inability to mount an immune response.

Figure 8
figure 8

Sparc-null mice have a greatly attenuated response to LPS. LPS was injected into the footpad of Sparc-null (Null) and Sparc WT (WT) mice. Measurements were taken at time points of 0, 24, 48, 72 and 96 h and the change in footpad swelling from the 0 time point is presented. *Significant differences between Sparc WT LPS versus PBS treatment. **Significant differences between Sparc WT versus Sparc-null response to LPS treatment, n=5/genotype/treatment group.

Full size image

Discussion

Sparc-null and WT mice have been used to assess differences in tumor development in the absence or presence of Sparc. The influence of the host immune cells on ECM production is reported to play a potential role in modulating the access of the tumor to adjacent tissue. However, increased access by immune cells does not necessarily mean that the immune cells are competent and capable of mounting a proper response. In this report, we demonstrate that Sparc-null mice have an altered immune system and demonstrate a lack of immune response to challenge. Major differences were observed in spleen morphology and in splenic immune cell populations.

With respect to the spleen, gross observation indicated that the spleens were larger in the Sparc-null mice. This was not due to a difference in the overall size of the mice, as the total body weights of Sparc-null and WT animals were similar, in agreement with published data.4 Furthermore, the spleen to body weight ratios were significantly higher in the Sparc-null mice, suggesting a specific difference in the spleens. The increase in size was also not due to an increase in edema, as the wet weight and dry weight ratios remained the same.

These data suggested that Sparc might play a role in the spleen. We found that Sparc is expressed in the spleen in endothelial cells within the white pulp, in the sinusoids of the perifollicular compartment, and in scattered cells throughout the white and red pulp and marginal zones. Although our data disagree with a previous report that found no Sparc expression within the spleen,25 the difference in results could be due to different fixations, antibodies and/or staining procedures. Our immunohistochemistry results are in agreement with our Western blot analysis, which also demonstrated Sparc in WT spleens. Furthermore, a study by Shen et al.26 also found Sparc expression in the stromal compartment of the spleen of normal mice. Thus, we propose that the loss of Sparc could alter overall spleen development and growth, and/or affect lymphocyte trafficking through the spleen. Recent reports indicate that SPARC is a ligand for Stabilin-1,27 a receptor present on macrophages and the noncontinuous sinusoidal cells in the spleen,28 and a ligand for VCAM-1 that mediates leukocyte transmigration.29 These studies support our contentions that the loss of Sparc could impact splenic development or alter immune responses.

Indeed, the morphological and immunohistochemical assessments resulted in two major observations. The first is that the loss of Sparc expression correlates with increased splenic white pulp and follicular lymphoid hyperplasia. These changes are consistent with a heightened humoral immune response, possibly due to response to a foreign or self-antigen. In addition, the fold increase in proliferation, as determined by Ki-67 detection, was sufficient to account for the fold increase by weight.

The second major observation is that there is a decrease of marginal zone cell layers and IgM-positive marginal zone B cells in the spleens of Sparc-null mice. In normal mice, immature, bone marrow-derived B cells in the arterial circulation can enter the spleen and flow through the splenic arterioles into the marginal sinuses. Homing chemokines can draw the B cells into the follicles where some differentiate into follicular B cells and others into marginal zone B cells. The follicular B cells can recirculate or remain in the splenic follicles ready to effect a T-cell-dependent humoral immune response to circulating antigens. Marginal zone B cells traffic to and localize in the marginal zone. Marginal zone stromal cell–B cell and B cell integrin–ECM interactions are believed to play a role in marginal zone B-cell localization;30, 31 however, the exact mechanisms that govern this process are not entirely known. Once in the marginal zone, marginal zone B cells are strategically positioned to mount a thymus-independent type 2 antigen response to circulating antigens such as bacterial capsular polysaccharide, and to transport circulating immune complexes into the splenic follicles and activate a T-cell-dependent follicular immune response. To address the role of Sparc in B-cell differentiation and trafficking, MOMA-1 staining of the MMM was used to identify the follicle/marginal zone boundary.30 The MOMA-1 staining pattern appeared normal for both WT and null spleens, suggesting that the loss of Sparc had no impact on the localization of these cells. However, the reduction of IgM-positive marginal zone B cells, along with the hyperplasia of white pulp in the Sparc-null mice, suggests several possible consequences requiring further investigation. The lack of Sparc may alter normal B-cell differentiation, prevent proper tracking of the marginal zone B cells into the marginal zone, contribute to poor retention of marginal zone B cells within the marginal zone, and/or promote activation of follicular and marginal zone B cells, and extramarginal zone migration of the latter.

Abnormal splenic architecture has been observed with other knockout mice. For instance, the spleens of Aiolos-null mice also have follicular hyperplasia and absent marginal zones,32 and those of Pyk-2-deficient mice lack marginal zones.33 In these examples, the underlying defects are thought to be intrinsic to the B cell. However, as the B cells do not appear to express Sparc at latter stages of development, we propose that it is the loss of Sparc in the sinusoidal and/or stromal cells that reduces marginal zone formation in Sparc-null mice. Given that Sparc influences ECM production and integrin-ECM interactions,34 and is a ligand for receptors present on splenic macrophages and sinusoidal cells,28 its loss could impact B-cell localization and retention in the marginal zone.

The Sparc-null mice appear to have slightly lower WBC counts and neutrophil counts in peripheral blood and a decreased proportion of CD19+ B cells together with an increased proportion of CD3+ T cells in bone marrow than their WT counterparts. However, consistent imbalances in blood and bone marrow WBC or lymphoid subsets were not identified in the Sparc-null mice. Nevertheless, a primary defect in lymphocyte production cannot be completely ruled out in these mice. Interestingly, it has been reported that Sparc is a stromal cell product that interacts with precursor B cells,35 and it is conceivable that the loss of Sparc in the bone marrow compartment could adversely influence early B-cell development. Recent reports do, in fact, support a role for Sparc as a differentiation factor where it is implicated in early myocardial cell differentiation36 and adrenal development.37

Having observed alterations in the T and B cells in the bone marrow population, and hyperplasia and altered marginal zone B cells in the spleens, we used the footpad model that could be used to assess both the innate and adaptive immune response. We found the Sparc-null mice could not even mount an innate response. It is of interest that in normal mice, circulating polysaccharide antigen can induce an early T-cell-independent humoral immune response mediated by splenic marginal zone B cells.30, 31 However, in this model the LPS is not circulating. Thus, the lack of immune response at this early stage suggests other immune impairments, in addition to the observed alterations.

In conclusion, these data suggest that Sparc may play an integral role in normal development and function of the murine immune system, and that the function of Sparc in this regard should be investigated further. Importantly, the data also indicate that Sparc-null mice with a C57BL/6 genetic background may not provide a suitable model for the assessment of the immune response to tumor, given that these mice have altered immune systems.

Materials and methods

Mice and genotyping

Sparc-null mice on a mixed 129SV/C57BL/6 background were originally obtained from Dr Chin Howe at the Wistar Institute, Philadelphia, PA, USA. The nulls were backcrossed with B6129SF1/J (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), which retained the C57BL/6 background. Heterozygotes were obtained to derive the Sparc WT (WT) controls and Sparc-nulls (null). Mice from 2 to 11 months of age were used: 2- to 3-month-old mice were used for morphology, weights, blood smear, automated blood analysis, flow and footpad challenge, 6-month-old mice were also used for flow, and 10- to 11-month-old mice were used for morphology, Ki-67, marginal zone, MOMA-1 and IgM analyses. Mice were age matched within all experiments.

Genotyping was assessed using PCR with 0.2 μ M primers including the Sparc forward (5′–3′) GAT GAG GGT GGT CTG GCC CAG CCC TAG ATG CCC CTC AC; Sparc reverse (5′–3′) CAC CCA CAC AGC TGG GGG TGA TCC AGA TAA GCC AAG; and neomycin reverse (5′–3′) GTT GTG CCC AGT CAT AGC CGA ATA GCC TCT CCA CCC AAG. PCR reactions were performed with 100 ng of mouse genomic DNA, to give a total of 25 μl, including the PCR buffer (10 mM Tris-HCl (pH-9.0), 50 mM KCl, 2.5 mM MgCl2 and 0.1% Triton X-100) containing 0.5 U of Taq DNA polymerase and 0.2 mM deoxynucleoside triphosphates. The reaction was carried out with an initial 1 min at 96°C followed by 40 cycles of denaturation at 96°C for 45 s, annealing at 70°C for 45 s, extension at 72°C for 6 min, ending with 72°C for 10 min and a hold at 4°C The PCR fragments are approximately 300 bp for the WT allele and between 500–600 bp for the null alleles.

Western blot analysis of Sparc in WT and null spleens

Protein was isolated using a single detergent lysis buffer as reported.14 Protein concentration was determined using the BCA protein assay kit (Pierce Chemical, Rockford, IL, USA). Samples (30 μg) were evaluated by Western blotting as previously reported,14 except using anti-mouse Sparc antibody (catalog no. AF942, R&D Systems, Minneapolis, MN, USA). Actin detection (1:500, no. sc-1616 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)) was used as a loading control.

Wet weights and dry weights

All animal experiments were conducted under approved IACUC protocols. Blood was collected for the blood smear analyses (see later), and then immune and non-immune organs (spleen, thymus, kidneys, liver and heart) were removed. The organs were weighed immediately for wet weight, dried overnight and weighed the next morning for dry weight (n=17 null; n=11 WT; of these n=14 male subjects; n=14 female subjects).

Morphological assessment of organs

Mice were perfused with saline followed by 10% formalin. The organs were removed, weighed and placed in vials containing 10% neutral buffered formalin. Formalin-fixed tissues were embedded in paraffin, sectioned (5 μm) and H&E stained for histomorphological assessment by a hematopathologist (RCH) and a neuropathologist (JAG) (n=8/group). For frozen tissues, mice were euthanized and the spleens were immediately snap frozen by liquid nitrogen.

Immunohistochemistry

All reagents are from Biocare Medical (Concord, CA, USA) unless otherwise noted and all incubations were at room temperature. Formalin-fixed, paraffin embedded 4-μm sections were dried in a 60°C oven for 1 h, deparaffinized to water and treated with a peroxidase block. Heat-induced antigen retrieval was used for both Sparc and Ki-67, using Diva in a pressure cooker or citrate buffer (pH 6.0) on a hotplate, respectively. Slides for CD31 were treated with trypsin for 15 min at room temperature. Immunohistochemistry was performed on the Biocare Nemesis 7200 autostainer. Sections were blocked in Sniper for 7 min and then incubated in 1:200 anti-Sparc (R&D Systems, no. AF942) for 45 min, or 1:250 Ki-67 (Dako (Carpinteria, CA, USA) no. M7249) for 40 min or CD31 (Biocare CM 303) for 3 h. Detection for Sparc was biotinylated anti-goat (Vector Laboratories (Burlingame, CA, USA) no. BA5000), for Ki-67 was biotinylated anti-rat (Vector Labs no. BA4001) followed by 4+ horseradish peroxidase (HRP) and Betazoid diaminobenzidine (DAB). CD31 detection was Rat on Mouse HRP Polymer per the manufacturer's instructions, followed by Betazoid DAB. All sections were briefly counterstained in CAT hematoxylin (n=4/genotype).

Snap frozen spleens were cut at 8-μm and the sections were picked up onto charged slides. They were air dried at room temperature overnight. They were fixed with two cycles of acetone for 10 min and air drying for 20 min. All reagents used, except for the antibodies, were purchased from Biocare Medical (Concord, CA, USA) and all washes were with Tris-buffered saline with triton (TBST). The procedure was carried out manually. Sections were incubated with Sniper block for 10 min, followed by incubation with rat anti-mouse MOMA-1 (1:80 in green diluent, Serotec (Raleigh, NC, USA) MCA947) at room temperature for 1 h. After a TBST rinse, the sections were incubated with anti-rat polymer-HRP 2 component detection per the manufacturer's instructions, and then incubated with Betazoid DAB for 4 min. Sections were then placed in denature solution (1:1 dilution) for 1.5 min, rinsed in TBST, and then exposed to DAB Sparkle enhancer for 1 min. After a TBST rinse, the sections were incubated with rat anti-mouse IgM (Southern Biotech (Birmingham, AL, USA) 1140-01) diluted 1:3000 in a 1:1 mix of green diluent and Sniper block for 50 min. After a TBST rinse, the sections were then incubated with anti-rat polymer-Alk Phos 2 component detection per instructions. The sections were given a TBST rinse, followed by Ferangi Blue chromogen for 10 min. Sections were mounted after counterstaining in Nuclear Fast Red for 15 s (n=3/group).

Quantitation of proliferative germinal center B cells

For each spleen, Ki-67-positive cells were manually counted in the germinal centers or red pulp regions over the entire section. The highest 10 counts for each compartment were averaged for each section. The average number of Ki-67-positive cells was compared between WT and null spleens (n=4/group).

Quantitation of marginal zone cell layers

For each spleen, five representative follicles were imaged. For each follicle, the marginal zone cell layers surrounding the follicle were counted manually. For each follicle, the cell layers in 2–3 separate regions were averaged. The counts per follicle for the five representative follicles were then averaged for each spleen. The average number of marginal zone cell layers was compared between WT and null spleens (n=4/group).

Quantification of white pulp

For each spleen, × 40 magnification images were captured. The total area and the areas of the white pulp were measured using Pro-Cite image analysis software. For each spleen, the area for the white pulp was calculated as a percentage of the total area. The percentages for each spleen/genotype were then averaged. The average percent area for white pulp was compared between WT and null spleens (n=4/group).

Imaging

Immunohistological images were taken at room temperature using a Nikon Eclipse E800M microscope with × 10, × 20 and × 40 objectives connected to a Nikon DXM1200C digital camera, and digitized using ACT-1C software on Dell Optiplex GX620 computers. Tiff images were imported into Adobe Photoshop for composite production. Insets are magnifications performed using Photoshop.

Blood smears and blood smear cell counts

Ethylenediaminetetraacetic acid anticoagulated peripheral blood (40 μl) was used to prepare smears following the wedge method. Four smears were prepared for each sample using precleaned super frost microscope slides (Fisher Scientific, Pittsburg, PA, USA). Smears were fixed by air drying before staining with TruColor Wright Stain (Beckman Coulter, Miami, FL, USA) following the manufacturer's instructions. A manual differential count of 100 WBCs was performed using the thin area of each slide. WBCs were classified as neutrophils, lymphocytes or monocytes based on morphological differences. The counts obtained for each set of four slides were analyzed using Excel software. The smears were reviewed by a hematopathologist (RCH) for morphological assessment (n=17 null; n=11 WT). The same animals were used for the wet and dry weight assessments.

Hematologic analyses

Mice were anesthetized for orbital blood collection. Blood was collected directly into 3.8% sodium citrate tubes (1:9 ratio of citrate to blood) and inverted immediately. Basophil, eosinophil, neutrophil, hematocrit, hemoglobin, lymphocyte, monocyte, platelet, WBC and red blood cell numbers were counted from whole blood using ABX Pentra 60C+ (Horiba ABX-USA, Irvine, CA, USA). Data from three experiments were combined for a total n=26 null and a total n=30 WT mice (experiment no. 1 n=10 null, n=12 WT; experiment no. 2 n=6 null, n=8 WT; experiment no. 3 n=10 null, n=10 WT).

Analysis of immune components

Peripheral blood, spleens and bone marrow were collected for isolation of lymphocytes. Cells were isolated by Ficoll–Hypaque (Amersham Biosciences, Piscataway, NJ, USA) centrifugation separation as described previously.38 Following this, the primary antibodies CD3 (Santa Cruz Biotechnology), CD19 (GeneTex Inc., San Antonia, TX, USA), CD4, CD8 (Serotec), CD45RA (BDPharmingen, San Jose, CA, USA), CD44, CD11c, CD14 and CD69 (eBioscience, San Diego CA, USA) were used singly or in combination as follows: CD3, CD4, CD8, CD44, CD3/CD44, CD19, CD19/CD44, CD11C, CD3+/CD8−/CD11C+, CD45RA, CD45RAHI, CD45RAMID, CD3/CD45RA, CD3+/CD45RAHI, CD3+/CD45RAMID, CD19/CD45RA, CD19+/CD45RAHI, CD19+/CD45RAMID, CD3+/CD44HI, CD3+/CD44MID, CD19+/CD44HI, CD19+/CD44MID. Secondary antibodies used were fluorescein isothiocyanate (FITC) anti-rabbit, AlexaFLuor 647 anti-rat (Invitrogen, Carlsbad, CA, USA), FITC anti-rat or PE anti-rat (Vector Laboratories, Burlingame, CA, USA). Controls were stained with secondary combinations only. A total of 104 events, gated for lymphocytes using forward and side scatter profiles were collected using a FacsCaliber (BDPharmingen) flow cytometer and analyzed using CellQuest software (n=10 for each genotype; n=5 for each gender with a genotype).

Footpad swelling response

Sparc WT and null mice were injected with 2.5 μg LPS (Sigma Co, St Louis, MO, USA) or sterile PBS alone. Footpad dimension was measured before and every 24 h for 4 days. Degree of swelling is given in mm as the difference between swelling responses at time measured versus original measurement before injection (n=5/genotype/group).

Statistical analyses

Two sample t-tests were used to compare differences between Sparc-null and WT mice for animal weights, organ wet and dry weights, spleen-to-whole body weight ratios, blood smear analyses, Ki-67 proliferation, white pulp and marginal zone cell layer measurements, and cell marker (blood, bone marrow and spleen) measurements. Generalized estimating equation methods were used to compare the two groups for hematologic assessment. These equations take into account that three separate experiments were conducted for hematologic outcomes. For the footpad analyses, Student's t-tests and Wilcoxon tests were carried out to compare the groups within genotypes and genotypes within groups. Comparisons were made for all animals, and in some cases for male subjects and female subjects separately. The testing level was set at 0.05 for all comparisons.

References

  1. Gilmour DT, Lyon GJ, Carlton MB, Sanes JR, Cunningham JM, Anderson JR et al. Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J 1998; 17: 1860–1870.

    CAS  Article  Google Scholar 

  2. Norose K, Clark JI, Syed NA, Basu A, Heber-Katz E, Sage EH et al. SPARC deficiency leads to early-onset cataractogenesis. Invest Ophthalmol Vis Sci 1998; 39: 2674–2680.

    CAS  PubMed  Google Scholar 

  3. Delany AM, Kalajzic I, Bradshaw AD, Sage EH, Canalis E . Osteonectin-null mutation compromises osteoblast formation, maturation, and survival. Endocrinology 2003; 144: 2588–2596.

    CAS  Article  Google Scholar 

  4. Bradshaw AD, Graves DC, Motamed K, Sage EH . SPARC-null mice exhibit increased adiposity without significant differences in overall body weight. Proc Natl Acad Sci USA 2003; 100: 6045–6050.

    CAS  Article  Google Scholar 

  5. Bradshaw AD, Reed MJ, Sage EH . SPARC-null mice exhibit accelerated cutaneous wound closure. J Histochem Cytochem 2002; 50: 1–10. Erratum in: J Histochem Cytochem 2002; 50: 875.

    CAS  Article  Google Scholar 

  6. Puolakkainen PA, Brekken RA, Muneer S, Sage EH . Enhanced growth of pancreatic tumors in SPARC-null mice is associated with decreased deposition of extracellular matrix and reduced tumor cell apoptosis. Mol Cancer Res 2004; 2: 215–224.

    CAS  PubMed  Google Scholar 

  7. Bos TJ, Cohn SL, Kleinman HK, Murphy-Ulrich JE, Podhajcer OL, Rempel SA et al. International Hermelin brain tumor symposium on matricellular proteins in normal and cancer cell-matrix interactions. Matrix Biol 2004; 23: 63–69.

    CAS  Article  Google Scholar 

  8. Mok SC, Chan WY, Wong KK, Cheung KK, Lav CC, Ng SW et al. SPARC, an extracellular matrix protein with tumor-suppressing activity in human ovarian epithelial cells. Oncogene 1996; 12: 1895–1901.

    CAS  PubMed  Google Scholar 

  9. Said N, Motamed K . Absence of host-secreted protein acidic and rich in cysteine (SPARC) augments peritoneal ovarian carcinomatosis. Am J Pathol 2005; 167: 1739–1752.

    CAS  Article  Google Scholar 

  10. Yiu GK, Chan WY, Ng SW, Chan PS, Cheung KK, Berkowitz RS et al. SPARC (secreted protein acidic and rich in cysteine) induces apoptosis in ovarian cancer cells. Am J Pathol 2001; 159: 609–622.

    CAS  Article  Google Scholar 

  11. Chlenski A, Liu S, Crawford SE, Volpert OV, DeVries GH, Evangelista A et al. SPARC is a key Schwannian-derived inhibitor controlling neuroblastoma tumor angiogenesis. Cancer Res 2002; 62: 7357–7363.

    CAS  PubMed  Google Scholar 

  12. Chlenski A, Liu S, Baker LJ, Yang Q, Tian Y, Salwen HR et al. Neuroblastoma angiogenesis is inhibited with a folded synthetic molecule corresponding to the epidermal growth factor-like module of the follistatin domain of SPARC. Cancer Res 2004; 64: 7420–7425.

    CAS  Article  Google Scholar 

  13. Ledda F, Bravo AI, Adris S, Bover L, Mordoh J, Podhajcer OL . The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma. J Invest Dermatol 1997a; 108: 210–214.

    CAS  Article  Google Scholar 

  14. Rempel SA, Golembieski WA, Ge S, Lemke N, Elisevich K, Mikkelsen T et al. SPARC: a signal of astrocytic neoplastic transformation and reactive response in human primary and xenograft gliomas. J Neuropathol Exp Neurol 1998; 57: 1112–1121.

    CAS  Article  Google Scholar 

  15. Ledda MF, Adris S, Bravo AI, Kairiyama C, Bover L, Chernajovsky Y et al. Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells. Nat Med 1997a; 3: 171–176.

    CAS  Article  Google Scholar 

  16. Golembieski WA, Ge S, Nelson K, Mikkelsen T, Rempel SA . Increased SPARC expression promotes U87 glioblastoma invasion in vitro. Int J Dev Neurosci 1999; 17: 463–472.

    CAS  Article  Google Scholar 

  17. Schultz C, Lemke N, Ge S, Golembieski WA, Rempel SA . Secreted protein acidic and rich in cysteine promotes glioma invasion and delays tumor growth in vivo. Cancer Res 2002; 62: 6270–6277.

    CAS  PubMed  Google Scholar 

  18. Sangaletti S, Stoppacciaro A, Guiducci C, Torrisi MR, Columbo MP . Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma. J Exp Med 2003; 98: 1475–1485.

    Article  Google Scholar 

  19. Brekken RA, Puolakkainen P, Graves DC, Workman G, Lubkin SR, Sage EH . Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM. J Clin Invest 2003; 111: 487–495.

    CAS  Article  Google Scholar 

  20. Puolakkainen PA, Brekken RA, Muneer S, Sage EH . Enhanced growth of pancreatic tumors in SPARC-null mice is associated with decreased deposition of extracellular matrix and reduced tumor cell apoptosis. Mol Cancer Res 2004; 2: 215–224.

    CAS  PubMed  Google Scholar 

  21. Alvarez MJ, Prada F, Salvatierra E, Bravo AI, Lutzky VP, Carbone C et al. Secreted protein acidic and rich in cysteine produced by human melanoma cells modulates polymorphonuclear leukocyte recruitment and antitumor cytotoxic capacity. Cancer Res 2005; 65: 5123–5132.

    CAS  Article  Google Scholar 

  22. Savani RC, Zhou Z, Arguiri E, Wang S, Vu D, Howe CC et al. Bleomycin-induced pulmonary injury in mice deficient in SPARC. Am J Physiol Lung Cell Mol Physiol 2000; 279: L743–L750.

    CAS  Article  Google Scholar 

  23. Sangaletti S, Gioiosa L, Guiducci C, Rotta G, Rescigno M, Stoppacciaro A et al. Accelerated dendritic-cell migration and T-cell priming in SPARC-deficient mice. J Cell Science 2005; 118: 3685–3694.

    CAS  Article  Google Scholar 

  24. Aycock RL, Bradshaw AC, Sage EH, Starcher B . Development of UV-induced squamous cell carcinomas is suppressed in the absence of SPARC. J Invest Dermatol 2004; 123: 592–599.

    CAS  Article  Google Scholar 

  25. Sage EH, Vernon RB, Decker J, Funk S, Iruela-Arispe ML . Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J Histochem Cytochem 1989; 37: 819–829.

    CAS  Article  Google Scholar 

  26. Shen Y, Iqbal J, Xiao L, Lynch RC, Rosenwald A, Staudt LM et al. Distinct gene expression profiles in different B-cell compartments in human peripheral lymphoid organs. BMC Immunol 2004; 5: 20.

    Article  Google Scholar 

  27. Kzhyshkowska J, Gratchev A, Goerdt S . Stabilin-1, a homeostatic scavenger receptor with multiple functions. J Cell Mol Med 2006; 10: 635–649.

    CAS  Article  Google Scholar 

  28. Kzhyshkowska J, Workman G, Cardo-Vila M, Arap W, Pasqualini R, Gratchev A et al. Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC. J Immunol 2006; 176: 5825–5832.

    CAS  Article  Google Scholar 

  29. Kelly KA, Allport JR, Yu AM, Sinh S, Sage EH, Gerszten RE et al. SPARC is a VCAM-1 counter-ligand that mediates leukocyte transmigration. J Leukoc Biol December 18 (E-pub ahead of print).

  30. Pillai S, Cariappa A, Moran ST . Marginal zone B cells. Ann Rev Immunol 2005; 23: 161–196.

    CAS  Article  Google Scholar 

  31. Lopes-Carvalho T, Kearney JF . Development and selection of marginal zone B cells. Immunol Rev 2004; 197: 192–205.

    Article  Google Scholar 

  32. Cariappa A, Tang M, Parng C, Nebelitskiy E, Carroll M, Georgopoulos K et al. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 2001; 14: 603–615.

    CAS  Article  Google Scholar 

  33. Guinamard R, Okigaki M, Schlesinger J, Ravetch JV . Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nature Immunol 2000; 1: 31–36.

    CAS  Article  Google Scholar 

  34. Bradshaw AD, Sage EH . SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 2001; 107: 1049–1054.

    CAS  Article  Google Scholar 

  35. Oritani K, Kincade PW . Identification of stromal cell products that interact with pre-B cells. J Cell Biol 1996; 134: 771–782.

    CAS  Article  Google Scholar 

  36. Stary M, Pasteiner W, Summer A, Hrdina A, Eger A, Weitzer G et al. Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro. Exp Cell Res 2005; 310: 331–343.

    CAS  Article  Google Scholar 

  37. Ishimoto H, Ginzinger DG, Matsumoto T, Hattori Y, Furuya M, Minegishi K et al. Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development. J Clin Endocrin Metab 2003; 91: 3208–3214.

    Article  Google Scholar 

  38. Chi DS, Harris NS . A simple method for the isolation of murine peripheral blood lymphocytes. J Immunol Methods 1978; 19: 169–172.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Dr Chin Howe for the Sparc-null mice. We thank Dr Hynda K Kleinman for her thoughtful discussion. This work was supported in part by the NIH/NCI Grant CA086997 (SAR). We are grateful for the continued support of the Barbara Jane Levy and the Gayle Halperin Kahn Funds.

Author information

Affiliations

  1. Department of Neurosurgery, Barbara Jane Levy Laboratory of Molecular Neuro-Oncology, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, MI, USA

    S A Rempel, E Mouzon, N Lemke, C R Schultz & W Golembieski

  2. Department of Pathology, Henry Ford Hospital, Detroit, MI, USA

    R C Hawley & J A Gutiérrez

  3. Department of Neurosurgery, Gayle Halperin Kahn Laboratory of Viral Onco-Therapeutics, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, MI, USA

    K R Bobbitt & C G Miller

  4. Department of Biostatistics and Research Epidemiology, Henry Ford Hospital, Detroit, MI, USA

    L R Schultz

  5. NIDCR, NIH, Bethesda, MD, USA

    J Koblinski & S VanOsdol

Authors
  1. S A Rempel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R C Hawley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J A Gutiérrez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E Mouzon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K R Bobbitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. N Lemke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C R Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L R Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. W Golembieski
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. J Koblinski
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. S VanOsdol
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. C G Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S A Rempel.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rempel, S., Hawley, R., Gutiérrez, J. et al. Splenic and immune alterations of the Sparc-null mouse accompany a lack of immune response. Genes Immun 8, 262–274 (2007). https://doi.org/10.1038/sj.gene.6364388

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gene.6364388

Keywords

  • SPARC
  • Sparc-null mouse
  • immune cells
  • spleen
  • marginal zone

Further reading

Search

Advanced search

Quick links