The immune system is equipped with mechanisms that downregulate hyperinflammation to avoid collateral damage. We demonstrated recently that unprimed T cells downregulate macrophage TNF production through direct interaction with macrophages in the spleen during LPS endotoxemia. How T cell migration toward macrophages occurs upon LPS injection is still not clear. In this study, we demonstrate that secreted osteopontin (sOPN) plays a role in the T cell migration to initiate the suppression of hyperinflammation during endotoxemia. Osteopontin levels in splenic macrophages were upregulated 2 h after LPS treatment, whereas T cell migration toward macrophages was observed 3 h after treatment. Neutralization of sOPN and blockade of its receptor, integrin αv, significantly inhibited CD4+ T cell migration and increased susceptibility to endotoxemia. Our study demonstrates that the sOPN/integrin αv axis, which induces T cell chemotaxis toward macrophages, is critical for suppressing hyperinflammation during the first 3 h of endotoxemia.

Inflammatory responses triggered by proinflammatory cytokines, such as TNF, work to eliminate microbial pathogens from hosts during infection. However, prolonged or excessive inflammation is harmful. Although innate immune cells are equipped with intrinsic inhibitory mechanisms to negatively control inflammation in innate immunity (15), we (6) and other investigators (79) found that adaptive immune cells also suppress early innate inflammatory responses during endotoxemia or sepsis. In our previous study (6), we demonstrated that T cells, but not B cells, are recruited in the splenic red pulp to interact with F4/80+ red pulp macrophages (RPMs) and suppress macrophage TNF expression by a direct T cell–macrophage interaction during LPS endotoxemia (6). Red pulp in the spleen is rich in RPMs but has scarce T cells. Once the cell interaction occurs, CD40L on the T cell surface ligates CD40 on the macrophage cell surface to initiate anti-inflammatory responses during LPS endotoxemia (6). Because these responses occur before T cell priming, T cell cognate Ags are not necessary to achieve the suppression. This suggested that even unprimed T cells play a critical role in immune responses to protect hosts from collateral damage by hyperinflammation. In this study, we further demonstrated a molecular mechanism downstream of macrophage CD40, through which TNF expression by macrophages is downregulated (6). CD40 signaling in macrophages induces IRAK1 sumoylation and nuclear translocation in the presence of TRAF2 (6). Nuclear IRAK1 binds to the Il10 promoter in macrophages to induce the expression of IL-10, which reduces Tnfa mRNA stability to eventually downregulate TNF-α production by macrophage (6). However, it was not clear how T cells migrate to splenic red pulp upon LPS treatment to interact with macrophages.

Osteopontin (OPN) is a glycosylated protein that is expressed in various immune cells, including macrophages and dendritic cells (10). There are two isoforms of OPN: intracellular OPN (iOPN) and secreted OPN (sOPN) (11, 12). Both iOPN and sOPN are generally known to induce proinflammatory responses (12), but iOPN can inhibit hyperinflammation during LPS endotoxemia (6). Because of alternative translation initiation, the iOPN nascent protein does not have a signal sequence; as a result, iOPN localizes in the cytoplasm instead of being secreted (11). iOPN plays a role as an adaptor molecule in signaling pathways downstream of innate immune receptors, as well as in cell motility, cytoskeletal rearrangement, and mitosis (12). Actually, iOPN is essential for IRAK1 sumoylation upon CD40 signaling activation in macrophages (6), as mentioned above. In contrast to iOPN, sOPN is a secreted protein, and the majority of OPN studies focused on sOPN. sOPN is known to play a role in attracting immune cells (13). OPN contains a tripeptide Arg-Gly-Asp (RGD) integrin-binding motif; therefore, sOPN ligates integrins, such as αvβ3, αvβ1, αvβ5, αvβ6, and α4β1(1416). Integrins are involved in immune cell migration by mediating the rolling and firm adhesion process during an inflammatory response. In particular, integrin αv plays a critical role in the migration of CD4+ T cells in inflamed tissue (17).

In this study, we demonstrate that sOPN plays a critical role in initiating T cell recruitment for T cell–macrophage interactions in the spleen to inhibit hyperinflammation during the early stage of LPS endotoxemia. Integrin αv expressed on T cells detects sOPN to achieve T cell chemotaxis. RPMs were able to produce sOPN 2 h after LPS injection, whereas CD4+ T cells constitutively expressed integrin αv, suggesting that the upregulation of sOPN by macrophages initiates T cell migration. CD4+ T cell migration toward macrophages was significantly inhibited by either OPN-neutralizing Ab or integrin αv–blocking Ab. Inhibiting T cell migration toward macrophages by these Abs significantly increased the susceptibility for LPS endotoxemia. Therefore, production of sOPN during an early stage of endotoxemia is critical to protect hosts from TNF-α–mediated hyperinflammation.

C57BL/6 and C57BL/6 Spp1−/− mice were purchased from The Jackson Laboratory. Sex-matched (male or female) and age-matched (6–7 wk old) animals were used for all of the experiments. All mice were maintained in barrier facilities and used according to Duke University Institutional Guidelines. This study was approved by the Duke University Institutional Animal Care and Use Committee.

To induce endotoxemia, Escherichia coli LPS (serotype 055:B5; Sigma-Aldrich) resuspended in PBS was injected i.p. into mice (40 mg/kg). Some mice were treated i.p. with integrin αv Ab (50 μg/mouse; BioLegend) or OPN Ab (20 μg/mouse; AF808: R&D Systems) 1 h prior to or 4 h after LPS injection.

Tissue preparation, staining, and confocal analysis were performed as previously described (6). Brilliant Violet 421–conjugated CD4 Ab (BioLegend; 10043823), Alexa Fluor 647–conjugated F4/80 Ab (BioLegend; 123122), and OPN Ab (AKm2A1; Santa Cruz) were used for staining. CD4+ T cell numbers in the red pulp were evaluated in images from 5–10 spleen sections/mouse, using Fiji software, by independent, blinded investigators.

RPMs (F4/80+) and CD4+ T cells were isolated using MicroBeads from the spleen of naive mice or mice treated with LPS (40 mg/kg, i.p.). Total RNA was extracted from RPMs and CD4+ T cells (1 × 106 cells) with TRIzol reagent (Invitrogen). cDNA synthesis was performed using qScript cDNA SuperMix (Quanta). Quantitative PCR was performed using KAPA SYBR FAST (Kapa Biosystems) and a thermocycler (Eppendorf). Relative expression of quantitative products was determined using the ΔΔCt method, with Actb mRNA as an internal control. Primers used for amplification are listed in Supplemental Table I. To evaluate sOPN protein levels, RPMs (1 × 106 cells/ml) were cultured in RPMI 1640 complete medium without stimulation for 3 h, and their supernatants were analyzed by ELISA, as previously described (10). Briefly, wells were coated with OPN Ab (AF808; R&D Systems) in coating buffer (0.1 M sodium carbonate, pH 9.5). Wells were blocked with 2% FBS in PBS for 1 h at room temperature. Detection was performed with biotinylated OPN Ab (BAF808; R&D Systems) and a secondary detection Ab (avidin-HRP Ab; BD Bioscience).

CD4+ T cells were obtained from spleens of LPS-injected mice or from naive mice and subjected to chemotaxis assays, as previously described (18). Briefly, CD4+ T cells (106 cells/well) were plated in the upper chambers of a Transwell (5 μm pore; Corning Costar). RPMs were isolated 2 h after LPS injection or from naive mice, and were cultured in RPMI 1640 complete medium without any stimulation for 3 h. RPM culture supernatant was added to the lower chambers of the Transwell. T cells were incubated for 5 h at 37°C in Transwell culture. The numbers of T cells that migrated to the lower chamber were counted. OPN Ab (10 μg/ml) was added to the bottom chamber, and CD4+ T cells were preincubated with integrin αv Ab (10 μg/ml) or integrin α4 Ab (10 μg/ml; BioLegend) and plated in the upper chambers. The numbers of T cells that spontaneously migrated to the lower chamber (medium alone in the lower chamber) were subtracted from those in the test groups.

Statistical analyses for all figures, with the exception of survival studies, were performed using the Student t test. Survival studies were analyzed with the Gehan–Breslow–Wilcoxon test. Statistical significance was set as p < 0.05. All data showed a normal distribution and are expressed as mean ± SEM.

We reported previously that LPS i.p. injection caused CD4+ T cells to migrate into the splenic red pulp, where macrophages were abundant, to initiate T cell–macrophage interactions and to inhibit hyperinflammation (6). However, it was not known what caused T cells to migrate toward macrophages during LPS endotoxemia. T cell migration occurs at ∼3 h and subsides by 6 h (6). Because sOPN plays a role in attracting immune cells as a ligand of various integrins (13), we evaluated sOPN production during LPS endotoxemia. Serum OPN levels were increased 5-fold 2 h after i.p. LPS injection (Fig. 1A). Because LPS induces OPN expression in macrophages (19), we also evaluated OPN expression in RPMs. Levels of Spp1 (Opn) mRNA and sOPN peaked at 1 and 2 h after i.p. LPS injection, with increases of 3- and 5-fold (Fig. 1B, 1C), respectively. To confirm the in situ distribution of OPN in the spleen, we carried out immunohistochemical analyses. OPN staining in the red pulp, but not the T cell zone, was identified 2 h after LPS treatment (Fig. 1D). The data suggest that RPMs are a source of sOPN in the spleen.

FIGURE 1.

Upregulation of sOPN in splenic macrophages during LPS endotoxemia. (A) Serum OPN levels at the indicated time points after i.p. LPS injection (40 mg/kg mouse weight). (B) Spp1 mRNA levels in RPMs that were isolated, at the indicated time points, after i.p. LPS injection. (C) sOPN levels in supernatants from RPM culture. RPMs were isolated at the indicated time points after i.p. LPS injection and cultured for 3 h in RPMI 1640 complete medium before harvesting supernatants. (D) Histological sections of spleens, isolated from naive mice and LPS-injected mice (2 h after i.p. injection), were stained to detect OPN (cyan), CD4+ T cells (CD4; red), and RPMs (F4/80; green). All of the experiments are representatives from at least two similar experiments for each. Scale bars denote 100 μm. *p < 0.05.

FIGURE 1.

Upregulation of sOPN in splenic macrophages during LPS endotoxemia. (A) Serum OPN levels at the indicated time points after i.p. LPS injection (40 mg/kg mouse weight). (B) Spp1 mRNA levels in RPMs that were isolated, at the indicated time points, after i.p. LPS injection. (C) sOPN levels in supernatants from RPM culture. RPMs were isolated at the indicated time points after i.p. LPS injection and cultured for 3 h in RPMI 1640 complete medium before harvesting supernatants. (D) Histological sections of spleens, isolated from naive mice and LPS-injected mice (2 h after i.p. injection), were stained to detect OPN (cyan), CD4+ T cells (CD4; red), and RPMs (F4/80; green). All of the experiments are representatives from at least two similar experiments for each. Scale bars denote 100 μm. *p < 0.05.

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To confirm whether LPS-treated macrophages induce T cell migration via sOPN, we performed a Transwell migration assay. RPMs were isolated from naive mice or from mice 2 h after i.p. LPS injection and cultured for 3 h in culture medium alone. Culture supernatant from RPMs was added to the bottom chamber of a Transwell, and migration of CD4+ T cells in the upper chamber was evaluated. CD4+ T cell migration was enhanced through RPMs isolated from LPS-treated mice but not by inducing T cell migration ability per se (Fig. 2A). In addition, OPN neutralization in the bottom chamber abolished CD4+ T cell migration (Fig. 2A), suggesting that sOPN plays a critical role in attracting T cells.

FIGURE 2.

Requirement of sOPN for T cell migration and control of hyperinflammation during LPS endotoxemia. (A) Ex vivo migration assay. Splenic T cells and RPMs were isolated from naive mice or from mice that received LPS injection. OPN-neutralizing Ab was added to the bottom chambers in the injection 2 h before cell harvest. (B) Localization of CD4+ T cells (CD4; red) and RPMs (F4/80; green) in the spleen. Spleen was isolated from wild-type and Spp1−/− mice 3 h after i.p. LPS injection. Representative images (left and middle panels) and results of quantitative analysis (right panel) are shown. Scale bars, 100 μm. (C) Mice were treated i.p. with OPN Ab 1 h prior to LPS injection, and spleens were harvested 3 h after LPS injection. T cell numbers were enumerated in images of red pulp. Shown are average values of 10 sections/mouse from three mice. (D and E) LPS was injected i.p. into mice with (●) or without (○) OPN-neutralization Ab injected i.p. 1 h prior to LPS injection (n= 12). Serum TNF-α levels 6 h after injection (D) and survival (E). *p < 0.05 versus control mice. n.s., not significant.

FIGURE 2.

Requirement of sOPN for T cell migration and control of hyperinflammation during LPS endotoxemia. (A) Ex vivo migration assay. Splenic T cells and RPMs were isolated from naive mice or from mice that received LPS injection. OPN-neutralizing Ab was added to the bottom chambers in the injection 2 h before cell harvest. (B) Localization of CD4+ T cells (CD4; red) and RPMs (F4/80; green) in the spleen. Spleen was isolated from wild-type and Spp1−/− mice 3 h after i.p. LPS injection. Representative images (left and middle panels) and results of quantitative analysis (right panel) are shown. Scale bars, 100 μm. (C) Mice were treated i.p. with OPN Ab 1 h prior to LPS injection, and spleens were harvested 3 h after LPS injection. T cell numbers were enumerated in images of red pulp. Shown are average values of 10 sections/mouse from three mice. (D and E) LPS was injected i.p. into mice with (●) or without (○) OPN-neutralization Ab injected i.p. 1 h prior to LPS injection (n= 12). Serum TNF-α levels 6 h after injection (D) and survival (E). *p < 0.05 versus control mice. n.s., not significant.

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We next asked whether sOPN was required in T cell migration in vivo during LPS endotoxemia. Histological analysis showed that T cells in wild-type mice successfully migrated to the splenic red pulp 3 h after LPS injection, but T cell migration was significantly reduced in OPN-deficient (Spp1−/−) mice (Fig. 2B). Enumeration of T cell numbers in the red pulp confirmed the failure of T cell recruitment to the red pulp in Spp1−/− mice (Fig. 2B). A similar reduction in T cell numbers was observed with in vivo OPN-neutralizing Ab treatment (Fig. 2C). These findings suggest that sOPN is critical for T cell migration during LPS endotoxemia. Previously, we reported that the lack of T cell interaction with macrophages caused the upregulation of macrophage TNF production, which resulted in increased susceptibility to LPS endotoxemia (6). Indeed, mice treated with OPN-neutralizing Ab upregulated serum TNF-α levels 6 h after LPS treatment (Fig. 2D) and showed earlier mortality than control IgG-treated mice (Fig. 2E). These results suggested that OPN secreted by macrophages promotes T cells to achieve T cell–macrophage interaction in controlling hyperinflammation in LPS endotoxemia. Therefore, sOPN also plays a host-protective role in endotoxemia but through a distinct mechanism from that of iOPN (6).

Because integrins are sOPN receptors and involved in immune cell migration, we next evaluated the roles of integrin αv and α4, two major receptors of sOPN, in T cell migration during LPS endotoxemia. First, Itgav mRNA was constitutively expressed in splenic CD4+ T cells before and after LPS injection, but Itga4 mRNA levels decreased after LPS injection (Supplemental Fig. 1). Constitutive expression of integrin αv protein on the surface of CD4+ T cells was confirmed by flow cytometry (Supplemental Fig. 1). To evaluate the functional involvement of integrin αv and α4 in T cell migration, we first performed a Transwell migration assay. Blocking integrin αv, but not integrin α4, abolished CD4+ T cell migration toward RPMs (Fig. 3A), suggesting that integrin αv plays a critical role in T cell migration. We then treated mice with an integrin αv–blocking Ab at two time points: 1 h prior to LPS injection and 4 h after LPS injection (Fig. 3B, 3C). Pre-LPS treatment with Ab significantly reduced T cell numbers in the red pulp, whereas post-LPS treatment with Ab did not have any impact on T cell migration. The data suggest that integrin αv plays its role during a narrow window in the early stage of endotoxemia.

FIGURE 3.

Integrin αv on T cells is critical for T cell migration and resistance to endotoxemia. (A) Ex vivo T cell migration toward RPMs. T cells were pretreated with blocking Ab for integrin αv or α4. (BE) In vivo integrin αv Ab treatment during endotoxemia. Ab was administered i.p. either 1 h before or 4 h after LPS injection. (B, C) Spleens were harvested 6 h after LPS injection. (B) Representative images of spleen. Scale bars, 100 μm. (C) T cell enumeration in red pulp. (D) Serum TNF-α levels 6 h after LPS injection. (E) Mouse survival with (filled circle, triangle) or without (open circle) integrin αv Ab. At least five mice/group. *p < 0.05 versus control mice.

FIGURE 3.

Integrin αv on T cells is critical for T cell migration and resistance to endotoxemia. (A) Ex vivo T cell migration toward RPMs. T cells were pretreated with blocking Ab for integrin αv or α4. (BE) In vivo integrin αv Ab treatment during endotoxemia. Ab was administered i.p. either 1 h before or 4 h after LPS injection. (B, C) Spleens were harvested 6 h after LPS injection. (B) Representative images of spleen. Scale bars, 100 μm. (C) T cell enumeration in red pulp. (D) Serum TNF-α levels 6 h after LPS injection. (E) Mouse survival with (filled circle, triangle) or without (open circle) integrin αv Ab. At least five mice/group. *p < 0.05 versus control mice.

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Because direct interactions between T cells and macrophages downregulate macrophage TNF expression (6), inhibition of T cell migration toward macrophages by OPN neutralization increased TNF expression, resulting in increased susceptibility to endotoxemia (Fig. 2D, 2E). We sought to determine the impact of integrin αv, an OPN receptor, in endotoxemia. Ab-mediated blockade of αv integrin 1 h prior to LPS injection increased serum TNF-α levels and significantly increased the susceptibility to endotoxemia (Fig. 3D, 3E). In agreement with these data showing no impact of integrin αv blockade 4 h after LPS injection (Fig. 3B, 3C), integrin αv Ab treatment 4 h after LPS injection did not alter serum TNF levels or host susceptibility (Fig. 3D, 3E).

Our findings strongly suggest that integrin αv on the T cell surface contributes to T cell migration to macrophages to control hyperinflammation by endotoxemia. Because extracellular matrix, such as collagen and fibronectin, supports integrin αv–mediated T cell migration in inflamed tissues (17), extracellular matrix in spleen (20) may also support T cell migration in LPS endotoxemia. Single-nucleotide polymorphisms (SNPs) in the human Itgav locus were identified; they are associated with chronic hepatitis B infection (21), sickle cell disease (22), and rheumatoid arthritis (23). Although it is not clear whether the SNPs have an impact on integrin αv expression on T cells, the SNPs in Itgav may be either a risk factor or a protective factor for sepsis and endotoxemia.

In this study, we demonstrated that OPN secreted by splenic macrophages is detected by integrin αv on the T cell surface and attracts T cells toward macrophages at a very early stage (∼3 h after treatment) of LPS endotoxemia (Fig. 4). Our data do not rule out the involvement of sOPN produced other than by the spleen. However, RPMs per se are the most proximate and plausible source of sOPN for splenic T cell migration toward RPMs. We reported previously that T cell–macrophage interactions suppress macrophage TNF production through CD40 signaling, in which iOPN is involved (6). In contrast, sOPN is dispensable in the CD40-mediated downregulation of macrophage TNF expression (6). Together with the data from our previous study (6), we suggest distinct roles for sOPN and iOPN during the early stages of endotoxemia: sOPN works first to attract T cells to macrophages and then iOPN works within macrophages to downregulate their TNF expression (Fig. 4). OPN is largely known to induce proinflammatory responses. However, this study clearly demonstrated that OPN also functions to downregulate inflammation during the first several hours of endotoxemia, during which time iOPN and sOPN participate to control hyperinflammation.

FIGURE 4.

Roles of sOPN and iOPN during LPS endotoxemia. T cells and macrophages are localized in different zones in the spleen. OPN expression is upregulated by LPS in RPMs, and sOPN is secreted by LPS-stimulated RPMs in the first 3 h after LPS stimulation. sOPN is detected by integrin αv on the CD4+ T cell surface, and T cells start migrating toward macrophages. T cells then interact with macrophages to stimulate the macrophage CD40 signaling pathway, in which iOPN is essential for the downregulation of macrophage TNF production (6).

FIGURE 4.

Roles of sOPN and iOPN during LPS endotoxemia. T cells and macrophages are localized in different zones in the spleen. OPN expression is upregulated by LPS in RPMs, and sOPN is secreted by LPS-stimulated RPMs in the first 3 h after LPS stimulation. sOPN is detected by integrin αv on the CD4+ T cell surface, and T cells start migrating toward macrophages. T cells then interact with macrophages to stimulate the macrophage CD40 signaling pathway, in which iOPN is essential for the downregulation of macrophage TNF production (6).

Close modal

We thank Jason Ashe for technical help.

This work was supported by the National Institutes of Health (R01-AI088100 and R21-AI103584 to M.L.S.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

iOPN

intracellular OPN

OPN

osteopontin

RPM

red pulp macrophage

SNP

single-nucleotide polymorphism

sOPN

secreted OPN.

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The authors have no financial conflicts of interest.

Supplementary data