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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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Lung tissue Sema4A and Sema4D expression and their regulation by allergen and VEGF. (A) Formalin-fixed paraffin-embedded lung tissue sections (top microphotographs) were deparaffinized and immunohistochemistry was performed employing Abs for the intracellular portions of Sema4A and Sema4D molecules. Bottom photomicrographs show immunohistochemistry on frozen lung tissue sections obtained from VEGF tg mice and control WT mice being on DOX water for 7 days. Sema4A expression in PBS-treated lungs was mainly limited to macrophages and dendritic like-cells. Tissue Sema4A expression was upregulated with allergen treatment and VEGF exposure and detected on APC-like cells and smooth muscle cells (both cell types marked with red arrows). Inserts show high magnification fields (100x) with marker-positive cells. Sema4D expression was detected in cell bundle-like shapes (red arrows, inserts) in the lungs obtained from both PBS- and OVA-treated mice. OVA treatment did not significantly modulate tissue levels of Sema4D. (B) Sema4A was abundantly expressed on APC-like cells (inserts, red arrows) in lymphoid tissue of allergen-treated WT mice. (C) Soluble Sema4D protein (120kDa) was detected in lung tissue lysates obtained from OVA-treated mice. Whole Sema4A protein (150 kDa), dimer (>250 kDa), and weak soluble Sema4A (120 kDa) were detected in BAL fluids of OVA-challenged mice. Arrows point to the molecular weight protein standard reference bars.
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Figure 2: Lung tissue Sema4A and Sema4D expression and their regulation by allergen and VEGF. (A) Formalin-fixed paraffin-embedded lung tissue sections (top microphotographs) were deparaffinized and immunohistochemistry was performed employing Abs for the intracellular portions of Sema4A and Sema4D molecules. Bottom photomicrographs show immunohistochemistry on frozen lung tissue sections obtained from VEGF tg mice and control WT mice being on DOX water for 7 days. Sema4A expression in PBS-treated lungs was mainly limited to macrophages and dendritic like-cells. Tissue Sema4A expression was upregulated with allergen treatment and VEGF exposure and detected on APC-like cells and smooth muscle cells (both cell types marked with red arrows). Inserts show high magnification fields (100x) with marker-positive cells. Sema4D expression was detected in cell bundle-like shapes (red arrows, inserts) in the lungs obtained from both PBS- and OVA-treated mice. OVA treatment did not significantly modulate tissue levels of Sema4D. (B) Sema4A was abundantly expressed on APC-like cells (inserts, red arrows) in lymphoid tissue of allergen-treated WT mice. (C) Soluble Sema4D protein (120kDa) was detected in lung tissue lysates obtained from OVA-treated mice. Whole Sema4A protein (150 kDa), dimer (>250 kDa), and weak soluble Sema4A (120 kDa) were detected in BAL fluids of OVA-challenged mice. Arrows point to the molecular weight protein standard reference bars.

Mentions: Lung tissue immunohistochemistry with Ab for the intracellular portion of Sema4A has shown that significant numbers of cells with macrophage- and DC-like morphology were positive for Sema4A in the control PBS-treated mouse lungs (Figure 2 A). Sema4A expression in the lung was increased during allergen-induced inflammation due to the influx of the marker-positive cells (3.2 ± 0.9 vs 6.2 ± 1.4 cells/high power 100x field, p < 0.042, PBS- vs OVA-treated mice). Bronchial epithelial cells showed a weak Sema4A expression whereas underlining epithelium smooth muscle cells were strongly Sema4A-positive (Figure 2 A). Isotype control Ab did not stain the tested tissues (Additional files, Figure 1) indicating Ab specificity and a definite upregulation of Sema4A in the allergen-treated lungs. In contrast to its relatively low expression in the lung tissue, Sema4A was readily detected in the lymphoid tissue of allergen-treated WT mice (Figure 2 B) and its expression was restricted, as expected, to APC-like cells.


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)

Lung tissue Sema4A and Sema4D expression and their regulation by allergen and VEGF. (A) Formalin-fixed paraffin-embedded lung tissue sections (top microphotographs) were deparaffinized and immunohistochemistry was performed employing Abs for the intracellular portions of Sema4A and Sema4D molecules. Bottom photomicrographs show immunohistochemistry on frozen lung tissue sections obtained from VEGF tg mice and control WT mice being on DOX water for 7 days. Sema4A expression in PBS-treated lungs was mainly limited to macrophages and dendritic like-cells. Tissue Sema4A expression was upregulated with allergen treatment and VEGF exposure and detected on APC-like cells and smooth muscle cells (both cell types marked with red arrows). Inserts show high magnification fields (100x) with marker-positive cells. Sema4D expression was detected in cell bundle-like shapes (red arrows, inserts) in the lungs obtained from both PBS- and OVA-treated mice. OVA treatment did not significantly modulate tissue levels of Sema4D. (B) Sema4A was abundantly expressed on APC-like cells (inserts, red arrows) in lymphoid tissue of allergen-treated WT mice. (C) Soluble Sema4D protein (120kDa) was detected in lung tissue lysates obtained from OVA-treated mice. Whole Sema4A protein (150 kDa), dimer (>250 kDa), and weak soluble Sema4A (120 kDa) were detected in BAL fluids of OVA-challenged mice. Arrows point to the molecular weight protein standard reference bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118960&req=5

Figure 2: Lung tissue Sema4A and Sema4D expression and their regulation by allergen and VEGF. (A) Formalin-fixed paraffin-embedded lung tissue sections (top microphotographs) were deparaffinized and immunohistochemistry was performed employing Abs for the intracellular portions of Sema4A and Sema4D molecules. Bottom photomicrographs show immunohistochemistry on frozen lung tissue sections obtained from VEGF tg mice and control WT mice being on DOX water for 7 days. Sema4A expression in PBS-treated lungs was mainly limited to macrophages and dendritic like-cells. Tissue Sema4A expression was upregulated with allergen treatment and VEGF exposure and detected on APC-like cells and smooth muscle cells (both cell types marked with red arrows). Inserts show high magnification fields (100x) with marker-positive cells. Sema4D expression was detected in cell bundle-like shapes (red arrows, inserts) in the lungs obtained from both PBS- and OVA-treated mice. OVA treatment did not significantly modulate tissue levels of Sema4D. (B) Sema4A was abundantly expressed on APC-like cells (inserts, red arrows) in lymphoid tissue of allergen-treated WT mice. (C) Soluble Sema4D protein (120kDa) was detected in lung tissue lysates obtained from OVA-treated mice. Whole Sema4A protein (150 kDa), dimer (>250 kDa), and weak soluble Sema4A (120 kDa) were detected in BAL fluids of OVA-challenged mice. Arrows point to the molecular weight protein standard reference bars.
Mentions: Lung tissue immunohistochemistry with Ab for the intracellular portion of Sema4A has shown that significant numbers of cells with macrophage- and DC-like morphology were positive for Sema4A in the control PBS-treated mouse lungs (Figure 2 A). Sema4A expression in the lung was increased during allergen-induced inflammation due to the influx of the marker-positive cells (3.2 ± 0.9 vs 6.2 ± 1.4 cells/high power 100x field, p < 0.042, PBS- vs OVA-treated mice). Bronchial epithelial cells showed a weak Sema4A expression whereas underlining epithelium smooth muscle cells were strongly Sema4A-positive (Figure 2 A). Isotype control Ab did not stain the tested tissues (Additional files, Figure 1) indicating Ab specificity and a definite upregulation of Sema4A in the allergen-treated lungs. In contrast to its relatively low expression in the lung tissue, Sema4A was readily detected in the lymphoid tissue of allergen-treated WT mice (Figure 2 B) and its expression was restricted, as expected, to APC-like cells.

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