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Keywords:

  • Extravasation;
  • Inflammation;
  • Thy-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Human Thy-1 (CD90) has been shown to mediate adhesion of inflammatory cells to activated microvascular endothelial cells via interaction with Mac-1 (CD11b/CD18) in vitro. Since there are no data showing the physiological relevance of Thy-1 for the recruitment of inflammatory cells in vivo, different inflammation models were investigated in Thy-1-deficient mice and littermate controls. In thioglycollate-induced peritonitis, the number of neutrophils and monocytes was significantly diminished in Thy-1-deficient mice. During acute lung inflammation, the extravasation of eosinophils and monocytes into the lung was significantly reduced in Thy-1-deficient mice. Moreover, during chronic lung inflammation, the influx of eosinophils and monocytes was also strongly decreased. These effects were independent of Thy-1 expression on T cells, as shown by the transplantation of WT BM into the Thy-1-deficient mice. In spite of the strong Thy-1 expression on T cells in the chimeric mice, the extravasation of the inflammatory cells in these mice was significantly diminished, compared to control mice. Finally, the altered number and composition of infiltrating leukocytes in Thy-1-deficient mice modified the chemokine/cytokine and protease expression at the site of inflammation. In conclusion, Thy-1 is involved in the control of inflammatory cell recruitment and, thus, also in conditioning the inflammatory microenvironment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The recruitment of inflammatory cells to sites of inflammation plays an important role in the pathogenesis of several inflammatory diseases. Leukocyte adhesion to endothelial cells (ECs) follows a multistep process, including the capture of free leukocytes out of the blood stream, rolling, firm adhesion, and transendothelial diapedesis. The importance of several adhesion molecules in this series of events has been described previously 1. In ICAM-1-deficient mice, neutrophil recruitment was significantly reduced, but it was not completely blocked in a chemical peritonitis model or in a lipopolysaccharide (LPS)-induced airway inflammation model, indicating the involvement of additional adhesion molecules 2, 3. Furthermore, leukocyte recruitment in experimental colitis was not affected by blocking ICAM-1 or MadCAM, whereas the blocking of VCAM-1 resulted in a significant attenuation of colitis 4. Thus, under specific inflammatory conditions, certain adhesion molecules mediate adhesion and transmigration of leukocytes into the perivascular tissue.

Recently, human Thy-1 expressed on ECs was identified as an adhesion molecule mediating the binding of neutrophils and monocytes to activated microvascular ECs 5. Thy-1 is a highly glycosylated GPI-anchored surface protein and a member of the immunoglobulin superfamily 6, 7, 8. In humans, Thy-1 is expressed on ECs at sites of inflammation or in tumours whereas ECs do not express Thy-1 in healthy tissue 5, 9. Thy-1 is also expressed on fibroblasts, neurons, and a subpopulation of haematopoietic stem cells in humans. Mac-1 expressed on neutrophils and monocytes was identified as a counter receptor for Thy-1 10. Furthermore, Thy-1 provides not only the mechanical support for cell adhesion but also triggers neutrophil effector functions, such as the secretion of matrix metalloproteinases (MMP-9) and chemotactic factors (CXCL8) 10, 11.

Thy-1-deficient mice, originally described by Nosten-Bertrand, are viable 12. Due to the strong expression of Thy-1 on neuronal cells and T cells (TCs) in mice, previous studies in Thy-1-deficient mice were focused on the investigation of the nervous system and TC functions. In spite of the high expression of Thy-1 on neuronal cells, the neuronal development proved to be unaffected in Thy-1-deficient mice 13. The lack of Thy-1 compromised some aspects of the social behaviour and the regeneration of axons after injuries 13. Beissert et al. demonstrated an impaired cutaneous immune response in Thy-1-deficient mice and a reduced activation of TCs 14. Thy-1-deficient mice display an abnormal retinal development 15 and develop a more severe lung fibrosis after bleomycin treatment 16. Although Thy-1 was identified in vitro as an adhesion molecule for the binding of leukocytes to activated ECs, the involvement of Thy-1 in the recruitment of leukocytes at sites of inflammation has not been investigated so far. To elucidate the impact of Thy-1 in the inflammatory process, different inflammation models were compared in Thy-1-deficient and WT mice. Using a thioglycollate-induced peritonitis model and an acute as well as chronic lung inflammation model, we demonstrate that Thy-1 plays an important role in the control of leukocyte recruitment at sites of inflammation and in the conditioning of the inflammatory tissue microenvironment.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Thy-1 is expressed on ECs at sites of inflammation in WT mice

Because Thy-1 displays a species-specific expression pattern 6, 17, 18, we analysed Thy-1 expression on ECs at sites of inflammation in mice. In healthy lung or healthy peritoneal tissue, Thy-1 was only hardly detectable on few ECs (Fig. 1A and B). In contrast to humans, Thy-1 seems to be slightly expressed on resting ECs in mice. However, upon induction of inflammation, Thy-1 expression on ECs was massively enhanced (Fig. 1C–F). We found strong Thy-1 expression on ECs of WT mice during lung inflammation, induced by immunization with OVA (Fig. 1D and F). In addition, Thy-1 was expressed on ECs in peritoneal tissue upon induction of inflammation, induced by thioglycollate (Fig. 1C and E).

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Figure 1. Thy-1 is expressed on ECs at sites of inflammation in WT mice. Murine Thy-1 was detected in cryostat sections using the avidin–biotin technique (Red staining). (A) Healthy peritoneal tissue, (B) healthy lung, (C and E) peritoneal tissue 24 h upon application of thioglycollate, (D and F) lung sections from OVA-immunized mice. As control, sections were stained with isotype control antibody (G and H). Arrows indicate vessels. Sections were counterstained with hematoxylin. One representative example is shown. (bar=100 μm).

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Neutrophil and monocyte extravasation into the peritoneal cavity is mediated by Thy-1

The functional role of Thy-1 in mediating adhesion and transmigration of neutrophils and monocytes was studied in a thioglycollate-induced peritonitis model in Thy-1−/− mice and littermates.

Prior to induction of inflammation, the blood leukocyte count as well as the subset proportions in Thy-1−/− mice and control mice were similar (Table 1). Induction of inflammation by i.p. injection of thioglycollate induced strong recruitment of leukocytes into the peritoneal cavity (Fig. 2A), which peaked 24 h after injection. The number of emigrated leukocytes was significantly decreased at 6 and 24 h after the i.p. injection in Thy-1−/− mice, compared to WT littermates. At later time points, no significant differences in the influx of inflammatory cells into the peritoneal cavity in Thy-1−/− and WT mice were detected (Fig. 2A). Analysis of extravasated cells 24 h after thioglycollate injection revealed that the recruitment of neutrophils and monocytes was significantly reduced in Thy1−/− mice (Fig. 2B). Lymphocytes were only marginally detectable.

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Figure 2. Extravasation of neutrophils and monocytes is attenuated in Thy-1−/− mice. Recruitment of leukocytes was induced by i.p. injection of 3% thioglycollate solution in Thy-1−/− mice (grey bars) and control littermates (black bars). (A) Cell counts in peritoneal fluid were determined at 0, 6, 24, 48, and 96 h post injection. Data are mean+SD (n=4 animals per group); *p<0.02 (t-test) (B) Cells emigrated at 24 h post-injection were identified by differential staining. Data are mean+SD (n=7 animals per group), *p=0.002 (t-test). Peritoneal tissue of WT littermates and Thy-1−/− mice was analysed by immunohistochemical staining, (C and D) H&E staining, arrow indicates infiltrating cells, (E and F) isotype control antibody, (G and H) detection of myeloid cells by staining with an anti-CD11b antibody, (I and J) detection of macrophages by staining with anti-F4/80, (K and L) detection of granulocytes by anti-Gr-1. Arrows indicate positive cells. Sections were counterstained with hematoxylin. One representative example is shown. (bar=100 μm)

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Histological analysis of peritoneal tissue confirmed these data. In contrast to those in Thy-1−/− mice, inflammatory cells in WT mice could be observed by H&E staining (Fig. 2C and D). Using immunohistochemical staining, a clear infiltration of CD11b+ cells was detected in the peritoneal tissue of WT mice (Fig. 2G). In Thy1−/− mice the infiltration was significantly inhibited (Fig. 2H). Further analysis of these infiltrates revealed that F4/80+macrophages (Fig. 2I and J) and Gr-1+neutrophil granulocytes (Fig. 2K and L) were decreased in peritoneal tissue of Thy1−/− mice. Taken together, Thy-1 plays an important role in the recruitment of neutrophils and monocytes during thioglycollate-induced peritoneal inflammation.

Eosinophil and monocyte extravasation during lung inflammation is diminished in Thy-1−/− mice

To emphasize the general importance of Thy-1 for the control of leukocyte extravasation during inflammation, we chose a model of acute and chronic lung inflammation in Thy-1−/− mice and their WT littermates. Acute inflammation was induced by immunization with OVA, resulting in lung inflammation characterized by an increased infiltration of eosinophils into the lung 19. OVA challenge of WT as well as Thy-1−/− mice resulted in a significant increase in total cell counts in the broncheoalveolar lavage (BAL), as compared to alum-treated control animals (Fig. 3A). Differential staining revealed that mainly eosinophils had migrated into the lung (Fig. 3B). Neutrophils and lymphocytes were only rarely detectable in the BAL of all mice. Importantly, mice genetically deficient in Thy-1 showed a significant reduction of total cells and, accordingly, a significantly decreased number of eosinophils in the BAL fluid after OVA immunization in comparison to WT littermates (Fig. 3A and B). In addition, the number of macrophages was decreased in the BAL of Thy-1−/− mice. Consequently, infiltration of the lung with inflammatory cells was clearly reduced in Thy-1−/− mice shown by histological staining (Fig. 3C–F). Measurement of the thickness of the perivascular infiltrate confirmed the significant reduction of lung inflammation in Thy-1−/− mice, compared to WT littermates (Fig. 3G).

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Figure 3. Thy-1−/− mice show decreased influx of inflammatory cells in BAL during acute and chronic lung inflammation. (A–G) Thy-1+/+ littermates and Thy-1−/− mice were immunized i.p. with OVA (20 μg) adsorbed to 2 mg of an aqueous solution of aluminum hydroxide on days 1 and 14 and challenged with 20 μg OVA in normal saline i.n. on day 14–16 and 21–23 (WT OVA, Thy-1−/− OVA). As control, mice received Alum i.p. and normal saline i.n. (WT Alum, Thy-1−/− Alum). The animals were sacrificed by CO2 asphyxiation at day 25. The trachea was cannulated, and the right lung was lavaged three times with 400 μL PBS. Total cell number (A) and cell numbers of eosinophils, neutrophils, macrophages, and lymphocytes (B) in the BAL fluid were determined. Data are mean+SD of three independent experiments (n≥13 animals per group). *p=0.002 (t-test); ♯ p=0.003 (Mann–Whitney Rank sum test). (C–F) One representative lung section of WT Alum (C), Thy-1−/− Alum (D), WT OVA (E) and Thy-1−/− OVA (F) stained by H&E is shown. Arrows indicate perivascular infiltrate. (G) Thickness of perivascular infiltrate was calculated using BZ-9000E analyser software. Data are mean+SD of one experiment (n=5 animals per group). *p=0.029 (Mann–Whitney Rank sum test). (H–N) Immunization protocol was prolonged to day 72 by challenging twice per wk. Animals were sacrificed by CO2 asphyxiation at day 72. The trachea was cannulated, and the right lung was lavaged three times with 400 μL PBS. Total cell number (I) and differential staining of BAL fluid cells (J) are shown. Data are mean+SD of two independent experiments (n=7 animals per OVA immunized group, n=3 per non-immunized mice). *p=0.013 (t-test); p=0.001 (t-test). (K–N) One representative lung section of WT Alum (K), Thy-1−/− Alum (L), WT OVA (M) and Thy-1−/− OVA (N) stained by H&E is shown. Arrows indicate perivascular infiltrate. (H) Thickness of perivascular infiltrate was calculated using BZ-9000E analyser software. Data are mean+SD of two independent experiments (n=7 animals per group). *p=0.017 (t-test).

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Chronic lung inflammation is characterized by extravasation of monocytes, eosinophils, and lymphocytes 19. To induce chronic lung inflammation, immunization was prolonged until day 72 by i.n. challenge of the mice two times per wk. As shown in Fig. 3I, the total number of infiltrating cells was significantly enhanced upon immunization, in comparison to alum control mice (Fig. 3I). In accordance with the acute inflammation, the influx of total cells, eosinophils, and macrophages was reduced in Thy-1−/− mice (Fig. 3J). The reduced extravasation into the lung in Thy-1−/−, compared to WT littermates was confirmed by histological staining of the lung section (Fig. 3K–N) and the measurement of the thickness of the perivascular infiltrate (Fig. 3H).

To exclude effects due to the genetic background, we also performed the thioglycollate-induced peritonitis and the OVA-induced acute lung inflammation in Thy-1−/− mice on 129/Sv background and 129/Sv WT mice. Again, lack of Thy-1 significantly reduced the extravasation of neutrophils and monocytes (Supporting Information Fig. 1).

TCs are not involved in decreased influx of inflammatory cells into the lung in Thy-1−/− mice

Considering the high expression of Thy-1 on murine TCs and the pathogenic role of TCs in OVA-induced lung inflammation 20, 21, we tested whether the differences observed in Thy-1−/− mice, compared to WT mice, were merely due to the lack of Thy-1 on TCs. Because Thy-1 is expressed only by TCs and not by other haematopoietic cells, we focused on the expression of Thy-1 on TCs. Thus, we generated BM chimeras by the reconstitution of hematoablative conditioned Thy-1−/− mice with BM cells, derived from WT mice. The resulting chimeric mice expressed Thy-1 on 60–70% of TCs (Fig. 4A). In comparison, in WT mice all TCs expressed Thy-1 and in Thy-1−/− mice neither of the TCs (Fig. 4A). Other haematopoietic cells do not express Thy-1. By immunohistochemical staining, we confirmed Thy-1 expression on ECs derived from OVA-immunized WT mice (Fig. 4B) and a lack of Thy-1 expression on ECs in Thy-1−/− mice (Fig. 4D). Most importantly, Thy-1 was also not detectable on ECs in the lungs of chimeric mice, but several cells in the inflammatory infiltrate (most likely TCs) were Thy-1 positive (Fig. 4F).

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Figure 4. Characterization of Thy-1−/− chimeric mice. For hematoablative conditioning, Thy-1-deficient mice were total-body γ-irradiated with a single dose of 7.5 Gy. BM transplantation was performed 4 h later by i.v. infusion of 1.5×107 BM cells derived from WT mice. (A) After 6 wk spleen cells were analysed by flow cytometry. Cells were gated on forward scatter (FS) and side scatter (Log SS) and subsequently, CD3-FITC positive cells were gated. Then, Thy-1 expression was analysed on CD3-positive cells of WT mice, Thy-1−/− mice, and chimeric mice. The histograms show one representative experiment out of 5. Percentage of Thy-1-positive cells are indicated. Murine Thy-1 was detected in cryostat sections of lung sections from OVA-immunized WT mice (B), Thy-1−/− mice (D), and Thy-1−/− chimeric mice (F) using the avidin–biotin technique (red staining). As control, sections were stained with isotype control antibody (control) (C, E and G). Arrows indicate vessels, the arrow head points to Thy-1-positive T cells in chimeric mice. Sections were counterstained with hematoxylin. One representative example is shown. (bar=100 μm)

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To exclude any effects of the lack of Thy-1 on TCs on the control of the extravasation of eosinophils during acute inflammation, chimeric mice were immunized with OVA, according to the standard protocol. Thy-1−/− mice and WT mice were immunized as controls. As shown in Fig. 5A, the total number of inflammatory cells in the BAL was significantly diminished in Thy-1−/− mice as well as in chimera, compared to WT mice. Differential staining showed that the number of both eosinophils and macrophages in the BAL fluid was diminished in Thy-1−/− mice as well as in chimera, compared to WT mice (Fig. 5B). Thus, although Thy-1−/− BM chimera expressed Thy-1 on 70% of TCs and Thy-1−/− mice did not express Thy-1 on TCs, in both mice the extravasation of leukocytes, especially eosinophils, was significantly reduced, compared to the WT mice. These results confirm that the decreased infiltration of the lung in Thy-1−/− mice was not merely a consequence of the lack of Thy-1 expression on TCs.

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Figure 5. The decreased influx of leukocytes in Thy-1−/− mice is independent of Thy-1 expression on T cells. BM chimeras were generated as described in Fig 4. After 4 wk chimeric mice, WT mice and Thy-1−/− mice were immunized with OVA (20 μg) adsorbed to 2 mg of an aqueous solution of aluminum hydroxide on days 1 and 14 and challenged with 20 μg OVA in normal saline i.n. on days 14–16 and 21–23 (Thy-1−/− chimera OVA, WT OVA and Thy-1−/− OVA, respectively). Controls received Alum i.p. and normal saline i.n. (WT Alum, Thy-1−/− Alum or Thy-1−/− chimera Alum). At day 25 the right lung was lavaged three times with PBS. Total cell numbers (A) and differential staining of cells (B) in the BAL fluid were determined. Data are mean+SD of two independent experiments (n≥9 animals per group, non-immunized n≥4). *p<0.02 (t-test)

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Lack of Thy-1 alters the pattern of chemokines, ILs, and proteases

We have shown that Thy-1 is involved in the control of leukocyte recruitment during inflammation. Next, we ask whether Thy-1-dependent leukocyte extravasation during inflammation has further functional consequences, such as the release of chemokines, cytokines, and proteases by the leukocytes. To address this issue, BAL and peritoneal fluid of WT and Thy-1−/− mice were compared. Cytokine and chemokine expression in the BAL was analysed by a membrane-based cytokine/chemokine array. The array results represent the chemokine/cytokine profile of three different WT and Thy-1−/− mice, respectively (Fig. 6).

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Figure 6. The lack of Thy-1 alters cytokine/chemokine and protease pattern at sites of inflammation. (A) Chemokines/cytokine profiles in the BAL of three OVA-immunized Thy-1−/− mice and WT mice were analysed by a membrane-based array. The densitometric data were adjusted to negative and positive controls. The quotient of the signal from the BAL of WT mice and Thy-1−/− mice from each membrane hybridization is shown. Data are mean+SD of two independent experiments. (B) CCL3, CCL17, CCL24, IL-4, and IL-5 expressions were analysed by RT-PCR in freshly separated or 24 h cultivated human eosinophils (eos) and monocytes (mo) with or without TNF-α, respectively. Expression of rps-26 was used as control. ntc: non template control. Data show one representative experiment out of three independent experiments. MMP-9 levels in (C) the BAL of control mice (WT Alum, Thy-1−/− Alum) and OVA-immunized mice (WT OVA, Thy-1−/− OVA) and in (D) the peritoneal fluid of Thy-1+/+ and Thy1−/− mice were detected by ELISA. Data are mean+SD of two independent experiments (n≥5 animals per group, *p<0.02 (t-test); p=0.014 (Mann–Whitney Rank sum test).

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In the BAL of WT mice IL-4, IL-5, eotaxin-2 (CCL24), TARC (CCL17), and MIP-1α (CCL3) were augmented (quotient >1.25), compared to Thy-1−/− mice (Fig. 6A). Analysis of mRNA expression of CCL3, CCL17, CCL24, IL-4, and IL-5 by semi-quantitative PCR revealed that these mediators were expressed by eosinophils and monocytes (Fig. 6B). In peritoneal fluid of WT mice, eotaxin-2 was also enhanced twofold, compared to Thy-1−/− mice (data not shown). In addition, we quantified the amount of MMP-9 since it is an important protease for the degradation of basement membrane components and, thus, plays a critical role during the transmigration of cells through basement membranes. MMP-9 was analysed by ELISA in the BAL and peritoneal fluid of WT mice and Thy-1−/−mice. Induction of lung inflammation by OVA challenges upregulated MMP-9 in BAL (Fig. 6C). Indeed, a significant decrease of MMP-9 levels was seen in the BAL of Thy-1−/− mice, compared to WT mice (Fig. 6C). Confirming these results, MMP-9 was also significantly decreased in the peritoneal fluid of Thy-1−/− mice, compared to WT mice (Fig. 6D).

Taken together, the lack of Thy-1 reduced the extravasation of granulocytes and monocytes during inflammation. As a consequence, the liberation of important granulocyte/monocyte derived chemokines, cytokines, and MMP-9 was decreased in Thy−/− mice.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The interaction of leukocytes with EC adhesion molecules plays an essential role in the control of immune and inflammatory responses, including arteriosclerosis, rheumatoid arthritis, psoriasis, and asthma 22, 23. Recently, we described human Thy-1 as a novel cell adhesion molecule on activated EC 5. Human Thy-1 mediates the adhesion of neutrophils and monocytes to activated EC via the interaction with Mac-1 10. Several in vitro studies suggest the importance of Thy-1 expressed on activated ECs for the adhesion of leukocytes 10. However, until now, there were no data showing the relevance of this interaction for the emigration of leukocytes at sites of inflammation in vivo. In the present study, we demonstrate the importance of Thy-1 in the control of granulocyte and monocyte recruitment to sites of inflammation in different mouse models for the first time.

First, we have to point out the different expression patterns of Thy-1 in humans and mice. In humans, Thy-1 is constitutively expressed on fibroblasts, neuronal cells, a subpopulation of blood stem cells, and glomeruli cells 6, 8, 18, 24. In addition, activated microvascular ECs express Thy-1 25. Importantly, in humans neither thymocytes nor TCs express Thy-1 17. Remarkably, in mice thymocytes and TCs express high levels of Thy-1 20. Considering these differences between species, we tested, first, whether Thy-1 is expressed on activated ECs during inflammatory processes in mice. Indeed, as in humans Thy-1 is expressed on ECs in mice during inflammation as shown by the Thy-1 expression on ECs in an OVA-induced airway inflammation model, as well as in a peritoneal inflammation model, induced by thioglycollate. Since we could ensure that Thy-1 expression on murine ECs is similar to that in humans, we used Thy-1−/− mice to investigate the role of Thy-1 for the control of the extravasation of leukocytes.

Thy-1 has been shown to be involved in the adhesion of monocytes and neutrophils to activated human microvascular ECs 5, and thioglycollate induces a strong extravasation of neutrophils and monocytes 26. Therefore, we, first, studied the recruitment of leukocytes into the peritoneal cavitiy after the injection of thioglyclloate in Thy-1−/− mice and control littermates. Indeed, in Thy-1−/− mice, the recruitment of neutrophils and monocytes was significantly inhibited. The relevance of Thy-1 in the control of leukocyte extravasation at sites of inflammation was verified in a lung inflammation model. Mice were challenged with OVA, resulting in acute lung inflammation associated with an enhanced number of eosinophils in the BAL, compared to animals exposed with saline, which confirmed the data described by Polte et al. 27. During long-term exposure to the antigen, leading to a chronic lung inflammation, the number of eosinophils and monocytes were significantly upregulated. The lack of Thy-1 resulted in decreased infiltration of eosinophils and monocytes into the lung during acute as well as chronic inflammation, indicating a key role of Thy-1 in airway inflammation induced by OVA.

Thus, investigating different inflammation models in Thy-1−/− mice, we could prove the physiological relevance of Thy-1 in the control of the recruitment of leukocytes at sites of inflammation in vivo. Due to strong expression of Thy-1 on TCs in mice, Thy-1 was investigated previously in mouse models with respect to the role of Thy-1 for development and function of TCs 14, 28, 29. Beissert et al. showed that Thy-1 deficiency in mice led to reduced contact hypersensitivity responses and a decreased irritant dermatitis, which were suggested to be due to a defective fine tuning of TC functions 14. In the light of our data, the impaired cutaneous immune responses in Thy-1−/− mice might, in addition to affected TC responses, also be caused by the lack of Thy-1 as an adhesion receptor on EC, mediating the extravasation of leukocytes during inflammation.

Considering the high expression of Thy-1 on murine TCs 29, 14 and the pathogenic role of TCs in OVA-induced lung inflammation 21, we excluded that the reduced lung inflammation in Thy-1−/− mice was dependent of the different Thy-1 expression levels on TCs. In Thy-1 BM chimera, the Thy-1-expression was detectable on 70% of TCs. Although Thy-1−/− BM chimera expressed Thy-1 on TCs and Thy-1−/− mice did not, airway inflammation was similarly reduced in both. In addition, BM transfer did not result in the incorporation of Thy-1-positive EC progenitor cells into the vessels, as Thy-1 staining of lungs revealed that vessels did not express Thy-1 in the BM chimeras. Thus, we can conclude that reduced extravasation of eosinophils and monocytes during airway inflammation in Thy-1-deficient mice is independent of Thy-1 expression on TCs and relies on the Thy-1 expression on activated ECs. Gerwin et al. used the approach of generating BM chimera to exclude effects of TCs in an inflammation model in ICAM-2−/− mice. Accordingly, they also showed that the lack of ICAM-2 on ECs was responsible for the decreased eosinophil emigration during lung inflammation 30.

As expected, the infiltration of leukocytes to the BAL fluid or into the peritoneal cavity was not completely inhibited in Thy-1−/− mice, suggesting a functional impact of further adhesion molecules. For example, ICAM-1−/− mice showed strongly decreased leukocyte infiltration in an OVA-induced inflammation model 31, as well as in a murine model of toluene diisocyanate-induced lung inflammation 32. ICAM-2−/− mice exhibited a delayed increase in eosinophil numbers in airway lumen and prolonged accumulation of eosinophils, compared to WT mice in an OVA immunization model 30. All these studies suggest a concerted action of several adhesion molecules during the recruitment of leukocytes to sites of inflammation. The present study demonstrates that Thy-1 is involved in the control of extravasation of leukocytes at sites of inflammation. While we did not define the steps of extravasation, which are controlled by Thy-1, our recent data do prove that Thy-1 mediates the adhesion of neutrophils and monocytes to activated ECs in vitro. Taken together, we suppose that Thy-1 is an alternate adhesion molecule on activated ECs, contributing to the control of leukocyte extravasation.

Finally, the lack of Thy-1 altered the number and composition of extravasated leukocytes, which led to changes of chemokine/cytokine and protease levels at inflammatory sites. Thus, reduced number of eosinophils and monocytes in the lung of Thy-1−/− mice was associated with decreased levels of MMP-9, eotaxin-2, IL-4, IL-5, TARC, and MIP-1α in BAL fluid. Moreover, MMP-9 and eotaxin-2 were decreased in the peritoneal cavity of Thy-1−/− mice upon induction of inflammation by thioglycollate. As shown by other groups, we also detected these products in granulocytes or monocytes by RT-PCR 33–37. Thus, the decreased number of granulocytes and macrophages in Thy-1−/− mice might be directly responsible for the reduced levels of these cytokines, chemokines, and protease in the BAL or peritoneal fluid of Thy-1−/− mice. Data from Furusho et al., describing an association of the number of eosinophils and the level of IL-4 and IL-5 concentrations in BAL in an murine model of toluene diisocyanate-induced asthma 32, support our findings. Our own PCR data and Watanabe et al. show that peripheral monocytes generate eotaxin-2 constitutively 35. Furthermore, IL-4 augmented eotaxin-2 expression in allergic lung inflammation 38. Thus, the indirect stimulation of chemokine/cytokine expression might also contribute to decreased levels of chemokines/cytokines in the BAL of Thy-1-deficient mice. For example, decreased levels of IL-4 in the BAL of Thy-1−/− mice might also add to the fact that eotaxin-2 is decreased in the BAL of Thy-1−/− mice. Moreover, we cannot exclude that interaction of granulocytes or monocytes with Thy-1 might also directly stimulate the secretion of the respective mediators. In fact, the interaction of neutrophils with Thy-1 directly stimulated MMP-9 release 11.

In conclusion, Thy-1 mediates the adhesion of granulocytes and monocytes to activated ECs and this interaction plays a pivotal role in the control of the emigration of granulocytes and monocytes from blood into peripheral tissue during inflammation. Consequently, the altered number and composition of extravasated leukocytes affect the inflammatory tissue microenvironment including the chemokine/cytokine and protease pattern.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Reagents and antibodies

Anti-mouse Thy-1 from BD Pharmingen (Heidelberg, Germany), anti-mouse F4/80 from Biolegend (San Diego, CA, USA), anti-mouse Thy-1 PE and anti-mouse CD3ε FITC, anti-mouse CD11b, anti-Gr-1 from Miltenyi Biotec (Bergisch Gladbach, Germany) were used.

Flow cytometry

Cells were harvested and washed twice in PBS. Then, 2×105 cells were incubated with indicated labelled antibody for 60 min at 4°C. After washing twice with PBS/Gelafusal (Serumwerke Bernburg, Germany)/sodium-acid, antibody binding was analysed by flow cytometry (FC 500, Beckman Coulter).

Immunhistochemistry

Cryostat sections were incubated with the antibodies indicated. Positive cells were identified by biotinylated goat anti-rat IgG and the avidin–biotin complex technique according to the manufacturer's protocol (supersensitive multilink alkaline phosphatase ready-to-use detection system, Biogenix, San Ramon, CA). The colour reaction of New Fuchsin substrate (DAKO, Hamburg, Germany) was used for detection of bound proteins. In control sections, primary antibodies were replaced with an isotype control antibody. Tissue sections were photographed using a DP70 CCD camera mounted on a BX41 light microscope (Olympus; Hamburg, Germany). Histological section were stained by H&E, photographed, and thickness of infiltrate was calculated using BZ-9000E analyzer software (Keyence BZ-9000E; Keyence, Neu-Isenburg; Germany).

Detection of cytokines and MMP-9

MMP-9 in the BAL and peritoneal fluid was measured by ELISA (R&D, Wiesbaden, Germany). A set of 48 cytokines/chemokines was detected by a membrane-based cytokine array according manufacture's protocol (RayBiotech, Norcross GA, USA). We used pooled BAL from two WT or two Thy-1−/− mice, respectively. The experiment was repeated with the BAL of a third mouse of each group. In summary, the array results represent the chemokine/cytokine profile of the BAL of three different WT and Thy-1−/− mice, respectively. The densitometric data were adjusted to negative and positive controls on the same membrane. Every chemokine/cytokine was detected by two different spots. The mean of the densitometric signal was used for evaluation. To identify differences in the amount of chemokine/cytokine the quotient of the signal from the BAL of WT mice and Thy-1−/− mice from each membrane hybridization was calculated. To get robust data, an increase of the signal was only accepted when the signal was enhanced over 25% (quotient >1.25) in both hybridizations.

Cell preparation

Human eosinophils were prepared from granulocytes upon Ficoll-density-gradient centrifugation of whole EDTA blood by depletion of CD16-positive neutrophils by magnetic separation according to manufacturer's protocol. Efficiency of separation was examined by anti-CD16 staining and flow cytometric analysis.

Human monocytes were separated from blood of healthy volunteers by magnetic cell separation using anti-CD14-beads (Miltenyi Biotec) as described previously 39.

RNA preparation and semi-quantitative PCR

Total RNA was isolated from human eosinophils or monocytes with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and 0.5 μg of total RNA were used for first strand cDNA synthesis with M-MLV reverse transcriptase (Promega, Madison, USA) according to the manufacturer's protocol. Semi-quantitative PCR was performed. The following primers (metabion, Martinsried, Germany) were used:

Ribosomal protein S26 (RPS26): forward: 5′-GCAGCAGTCAGGGACATTTCTG-3′, reverse: 5′-TTCACATACAGCTTGGGAAGCA-3′, CCL3: forward: 5′-ATGCAGGTCTCCACTGCTG-3′, reverse: 5′-TCGCTGACATATTTCTGGACC-3′, CCL17: forward: 5′-CTCGAGGGACCAATGTGG-3′, reverse: 5′-GACCTCTCAAGGCTTTGCAG-3′, CCL24: forward: 5′-GGTCATCCCCTCTCCCTG-3′, reverse: 5′-TAGCAGGTGGTTTGGTTGC-3′, IL-4: forward: 5′-ACAGCCACCATGAGAAGGAC-3′, reverse: 5′- TTTCCAACGTACTCTGGTTGG-3′, IL-5: forward: 5′- GAAAGAGACCTTGGCACTGC-3′, reverse: 5′- CCACTCGGTGTTCATTACACC-3′. Specifity of PCR products was verified by DNA sequencing.

Mice

Thy-1−/− mice were a kind gift of Prof. R. Morris, King's College London 12. Thy-1-deficient (Thy-1−/−) mice were established on a 129/Sv×C57BL/6 background as described previously 12. F2 littermates from the intercross of F1 Thy-1+/− mice were used for comparative studies between Thy-1−/− and Thy-1+/+ mice. Results were confirmed using Thy-1−/− and WT mice on 129/Sv background (Supporting Information Fig. 1). Mice were allowed food and water ad libitum, and kept under a 12-h light–dark cycle. All animal experiments were performed according to institutional and state guidelines. The Committee on Animal Welfare of Saxony approved animal protocols used in this study (TVV02/09). Blood cell counts and subset distribution were determined using Animal Blood Cell Counter (Scil Vet ).

Thy-1−/− chimeric mice were generated by irradiation of 6 wk old Thy-1−/− mice with 7.5 Gray. BM cells were collected from femora and tibiae of WT mice by flushing the opened bones with PBS/2.5% FCS. After centrifugation, the cells were washed three times with PBS. BM transplantation was performed by intravenous (i.v.) infusion of 1.5×107 BM cells per mouse into the tail vein of the Thy-1−/− recipients 4 h after irradiation. After a reconstitution time of 6 wk the immunization protocol was started. For controlling reconstitution splenic TCs were analysed for expression of Thy-1 by cytofluorometric analysis at day 25 of the immunization protocol.

Lung inflammation

Mice were immunized by a standard immunization protocol as described previously 27. In brief, mice were immunized with OVA (20 μg; Sigma-Aldrich, Steinheim, Germany) adsorbed to 2 mg of an aqueous solution of aluminium hydroxide and magnesium hydroxide (Perbio Science, Bonn, Germany) i.p. on days 1 and 14, followed by 20 μg OVA in 40 μL normal saline given i.n. on days 14–16, 21–23. Control mice received Alum i.p. and normal saline i.n. Mice were sacrificed on day 25.

To induce a chronic inflammation standard protocol was prolonged by OVA application until day 72 by administration of OVA i.n. twice per wk as described previously 19. Animals were sacrificed by CO2 asphyxiation. The trachea was cannulated, and the right lung was lavaged three times with 400 μL PBS. Cells in the lavage fluid were counted, and BAL cell differentials were determined on slide preparations stained with Diff Quik (Dade Behring). At least 100 cells were differentiated by light microscopy based on conventional morphologic criteria. Neutrophils displayed a multilobed nucleus and a fine pink staining. Eosinophils are characterized by the bilobed nucleus and deep pink staining of the cytoplasm. Lymphocytes have got a large, round, deeply blue nucleus. Monocytes were identified by the kidney shaped or bilobed nucleus.

Cell-free BAL supernatant was collected for cytokine and MMP-9 detection.

Thioglycollate-induced peritonitis

Mice were injected i.p. with 1 mL of 3% thioglycollate media (BD Biosciences) or PBS as control. At indicated time points peritoneal lavage was collected. Cells in the lavage fluid were counted and cell differentials were determined on slide preparations stained with Diff Quik (Dade Behring, Marburg, Germany). Cells were differentiated as described above. Cell-free peritoneal fluid was collected. Peritoneal tissue was dissected for histological studies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We greatly appreciate the technical assistance of Mr. Danny Gutknecht. We thank Manuela Ackermann for performing i.v. injection and Jutta Jahns for irradiation of mice. This work was supported by the Deutsche Forschungsgemeinschaft to Anja Saalbach and Ulf Anderegg (SA 683/2-1).

Conflict on interest: The authors declare no financial or commercial conflict of interest.

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  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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