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Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor.

Smith EP, Shanks K, Lipsky MM, DeTolla LJ, Keegan AD, Chapoval SP - BMC Immunol. (2011)

Bottom Line: Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins".We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes.CD72 was found on lung immune, inflammatory, and epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT

Background: Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined.

Results: Allergen treatment and lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly upregulated by allergen. Soluble Sema4D protein was present in the lung lysates and a whole Sema4A protein plus its dimer were readily detected in the bronchoalveolar (BAL) fluids under inflammation.

Conclusions: This study clearly shows that neuroimmune semaphorins Sema4A and Sema4D and their receptors might serve as potential markers for the allergic airway inflammatory diseases. Our current findings pave the way for further investigations of the role of immune semaphorins in inflammation and their use as potential therapeutic targets for the inflammatory lung conditions.

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Regulation of Sema4A and Sema4D expression on lung immune cells by allergen and VEGF. Flow cytometric detection of Sema4A and Sema4D expression on lung and spleen DC (A-B), lung T and B cells (C), and MHCII+ cells (D). Single cell suspensions were prepared as described in Methods. Conventional DC were identified by staining cells with anti-CD11c, -MHCII, and -CD11b Abs used for lung cDC detection. Highly fluorescent macrophages were gated out from the further analysis as large cells on FSC-SSC. (A) No cell surface Sema4A and low Sema4D expression was found on lung cDC. Spleen CD11c+MHCIIneg DC subset demonstrated high Sema4A and Sema4D expression which was further upregulated by allergen. (B) Intracellular Sema4A was targeted to a specific population of CD11cintermed/MHCIIlow cells under inflammatory conditions. (C-D) Low Sema4A and high Sema4D expression was detected on lung B220+ cells, CD3+ cells, and MHCII+ cells.
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Figure 3: Regulation of Sema4A and Sema4D expression on lung immune cells by allergen and VEGF. Flow cytometric detection of Sema4A and Sema4D expression on lung and spleen DC (A-B), lung T and B cells (C), and MHCII+ cells (D). Single cell suspensions were prepared as described in Methods. Conventional DC were identified by staining cells with anti-CD11c, -MHCII, and -CD11b Abs used for lung cDC detection. Highly fluorescent macrophages were gated out from the further analysis as large cells on FSC-SSC. (A) No cell surface Sema4A and low Sema4D expression was found on lung cDC. Spleen CD11c+MHCIIneg DC subset demonstrated high Sema4A and Sema4D expression which was further upregulated by allergen. (B) Intracellular Sema4A was targeted to a specific population of CD11cintermed/MHCIIlow cells under inflammatory conditions. (C-D) Low Sema4A and high Sema4D expression was detected on lung B220+ cells, CD3+ cells, and MHCII+ cells.

Mentions: Flow cytometry analysis of lung single cell suspensions has shown that, in contrast to lymphoid organs, there was no significant Sema4A expression on lung APC surface (Figure 3 A). This included lung cDC which were defined as CD11c+MHCIIlow cells in PBS-treated mice and as CD11c+/MHCII+ cells in OVA-treated mice [19]. Low number of lung plasmacytoid (p)DC, which were defined as CD11cintermedB220+ cells [19], were found to express Sema4A (from 7.5% to 17.9%, PBS vs OVA-treated mice, data not shown). Intracellular staining of the lung cells obtained by an enzymatic tissue digest has shown that Sema4A expression was predominantly targeted to a specific cell subset, namely CD11cintermed MHCIIlow cells (Figure 3 B) which could potentially include pDC, precursors of conventional lung DC, and a subset of lung macrophages [19,21,22]. Of note, neither CD11c+MHCII- cells nor CD11c-MHCII+ cells evaluated showed measurable levels of intracellular Sema4A (data not shown). Initial low level of lung cDC Sema4D expression was further downregulated after OVA treatment (Figure 3 A) possibly due to the previously described molecule shedding from the cell surface in inflammation [11,23]. Indeed, the presence of an extracellular portion of Sema4D (120 kB) in allergic but not control lungs was confirmed by Western blot employing tissue lysates (Figure 2 C).


Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor.

Smith EP, Shanks K, Lipsky MM, DeTolla LJ, Keegan AD, Chapoval SP - BMC Immunol. (2011)

Regulation of Sema4A and Sema4D expression on lung immune cells by allergen and VEGF. Flow cytometric detection of Sema4A and Sema4D expression on lung and spleen DC (A-B), lung T and B cells (C), and MHCII+ cells (D). Single cell suspensions were prepared as described in Methods. Conventional DC were identified by staining cells with anti-CD11c, -MHCII, and -CD11b Abs used for lung cDC detection. Highly fluorescent macrophages were gated out from the further analysis as large cells on FSC-SSC. (A) No cell surface Sema4A and low Sema4D expression was found on lung cDC. Spleen CD11c+MHCIIneg DC subset demonstrated high Sema4A and Sema4D expression which was further upregulated by allergen. (B) Intracellular Sema4A was targeted to a specific population of CD11cintermed/MHCIIlow cells under inflammatory conditions. (C-D) Low Sema4A and high Sema4D expression was detected on lung B220+ cells, CD3+ cells, and MHCII+ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Regulation of Sema4A and Sema4D expression on lung immune cells by allergen and VEGF. Flow cytometric detection of Sema4A and Sema4D expression on lung and spleen DC (A-B), lung T and B cells (C), and MHCII+ cells (D). Single cell suspensions were prepared as described in Methods. Conventional DC were identified by staining cells with anti-CD11c, -MHCII, and -CD11b Abs used for lung cDC detection. Highly fluorescent macrophages were gated out from the further analysis as large cells on FSC-SSC. (A) No cell surface Sema4A and low Sema4D expression was found on lung cDC. Spleen CD11c+MHCIIneg DC subset demonstrated high Sema4A and Sema4D expression which was further upregulated by allergen. (B) Intracellular Sema4A was targeted to a specific population of CD11cintermed/MHCIIlow cells under inflammatory conditions. (C-D) Low Sema4A and high Sema4D expression was detected on lung B220+ cells, CD3+ cells, and MHCII+ cells.
Mentions: Flow cytometry analysis of lung single cell suspensions has shown that, in contrast to lymphoid organs, there was no significant Sema4A expression on lung APC surface (Figure 3 A). This included lung cDC which were defined as CD11c+MHCIIlow cells in PBS-treated mice and as CD11c+/MHCII+ cells in OVA-treated mice [19]. Low number of lung plasmacytoid (p)DC, which were defined as CD11cintermedB220+ cells [19], were found to express Sema4A (from 7.5% to 17.9%, PBS vs OVA-treated mice, data not shown). Intracellular staining of the lung cells obtained by an enzymatic tissue digest has shown that Sema4A expression was predominantly targeted to a specific cell subset, namely CD11cintermed MHCIIlow cells (Figure 3 B) which could potentially include pDC, precursors of conventional lung DC, and a subset of lung macrophages [19,21,22]. Of note, neither CD11c+MHCII- cells nor CD11c-MHCII+ cells evaluated showed measurable levels of intracellular Sema4A (data not shown). Initial low level of lung cDC Sema4D expression was further downregulated after OVA treatment (Figure 3 A) possibly due to the previously described molecule shedding from the cell surface in inflammation [11,23]. Indeed, the presence of an extracellular portion of Sema4D (120 kB) in allergic but not control lungs was confirmed by Western blot employing tissue lysates (Figure 2 C).

Bottom Line: Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins".We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes.CD72 was found on lung immune, inflammatory, and epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

ABSTRACT

Background: Semaphorins were originally identified as molecules regulating a functional activity of axons in the nervous system. Sema4A and Sema4D were the first semaphorins found to be expressed on immune cells and were termed "immune semaphorins". It is known that Sema4A and Sema4D bind Tim-2 and CD72 expressed on leukocytes and PlexinD1 and B1 present on non-immune cells. These neuroimmune semaphorins and their receptors have been shown to play critical roles in many physiological and pathological processes including neuronal development, immune response regulation, cancer, autoimmune, cardiovascular, renal, and infectious diseases. However, the expression and regulation of Sema4A, Sema4D, and their receptors in normal and allergic lungs is undefined.

Results: Allergen treatment and lung-specific vascular endothelial growth factor (VEGF) expression induced asthma-like pathologies in the murine lungs. These experimental models of allergic airway inflammation were used for the expression analysis of immune semaphorins and their receptors employing immunohistochemistry and flow cytometry techniques. We found that besides accessory-like cells, Sema4A was also detected on bronchial epithelial and smooth muscle cells, whereas Sema4D expression was high on immune cells such as T and B lymphocytes. Surprisingly, under inflammation various cell types including macrophages, lymphocytes, and granulocytes in the lung expressed Tim-2, a previously defined marker for Th2 cells. CD72 was found on lung immune, inflammatory, and epithelial cells. Bronchial epithelial cells were positive for both plexins, whereas some endothelial cells selectively expressed Plexin D1. Plexin B1 expression was also detected on lung DC. Both allergen and VEGF upregulated the expression of neuroimmune semaphorins and their receptors in the lung tissue. However, the lung tissue Sema4A-Tim2 expression was rather weak, whereas Sema4D-CD72 ligand-receptor pair was vastly upregulated by allergen. Soluble Sema4D protein was present in the lung lysates and a whole Sema4A protein plus its dimer were readily detected in the bronchoalveolar (BAL) fluids under inflammation.

Conclusions: This study clearly shows that neuroimmune semaphorins Sema4A and Sema4D and their receptors might serve as potential markers for the allergic airway inflammatory diseases. Our current findings pave the way for further investigations of the role of immune semaphorins in inflammation and their use as potential therapeutic targets for the inflammatory lung conditions.

Show MeSH
Related in: MedlinePlus