Table 1.
Clinical characteristics of cases used in this study.
Fig 1.
NRPs expression in tissue-specific macrophages compared to immunostaining with a cocktail of anti-CD68 and anti-CD163 antibodies.
Tissue-specific macrophages were recognized by immunostaining with a cocktail of anti-CD68 and CD163 antibodies in serial sections. NRP-1 and NRP-2 expression was not observed in tissue-specific macrophages of brain (microglia), liver (Kupffer cells) and spleen (red pulp macrophages). Black arrows indicate the neuronal staining of NRPs in brain. Serial sections were counterstained with hematoxylin. NRP-1, neuropilin 1; NRP-2, neuropilin 2.
Fig 2.
NRPs expression in tissue-specific macrophages compared to immunostaining with a cocktail of anti-CD68 and anti-CD163 antibodies.
Tissue-specific macrophages were recognized by immunostaining with a cocktail of anti-CD68 and CD163 antibodies in serial sections. NRP-1 and NRP-2 expression was detected in alveolar macrophages in lung, but not in lymph node (sinus macrophages). And NRP-1 and NRP-2 also expressed on bronchial macrophages. Green arrow indicates NRP-2 expression on lymphatic vascular endothelium, used as positive control. Serial sections were counterstained with hematoxylin. NRP-1, neuropilin 1; NRP-2, neuropilin 2.
Fig 3.
NRPs expression from different sources in alveolar macrophages and, IHC with a cocktail of anti-CD68 and anti-CD163 antibodies in serial section of in-situ PCR.
(A) Immunohistochemistry showed alveolar macrophages expressing NRP-1 as detected by using 3 different antibodies from Abcam, Santa Cruz Biotechnology and Invitrogen. Serial sections were counterstained with hematoxylin. (B) Isotype or negative control showed no reactivity. It was counterstained with hematoxylin. (C) All alveolar macrophages showed positivity with CD68 and CD163. NRP-1, neuropilin 1.
Fig 4.
NRPs mRNAs expression in normal tissues (RT-PCR) and on alveolar macrophages in physiologically normal lung (in situ-PCR).
(A) By reverse transcriptase polymerase chain reaction (RT-PCR), NRP-1 mRNA was expressed in normal brain (lanes 1, 2), liver (lanes 3, 4), spleen (lanes 5, 6), lymph node (lanes 7, 8) and lung (lanes 9, 10 and 11). NRP-2 mRNA was expressed in normal lung and brain but was not expressed in liver, spleen and lymph node. N represents the negative control, and M represents the 20 base-pair DNA ladder. (B) NRP-1 and NRP-2 mRNAs of alveolar macrophages in physiologically normal lung (remote to the cancer nest), as observed by in situ-polymerase chain reaction (in situ-PCR). NRP-1, neuropilin 1; NRP-2, neuropilin 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig 5.
NRPs expression in intravascular macrophages in physiologically normal lung by immunostaining.
Intravascular macrophages (blood monocytes) were recognized by immunostaining with a cocktail of anti-CD68 and CD163 antibodies. NRP-1 and NRP-2 expression was observed in intravascular macrophages. Green arrow indicates NRP-1 expression on vascular endothelium, used as positive control. Serial sections were counterstained with hematoxylin. NRP-1, neuropilin 1; NRP-2, neuropilin 2.
Table 2.
Comparison of neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) expression in alveolar macrophages and interstitial macrophages in lung tissue remote to the cancer nest (physiologically normal lung) (n = 5).
Fig 6.
Immunohistochemistry of NRPs expression on alveolar macrophages in lung tissue (adjacent to the cancer margin, inflamed and physiologically normal).
NRP-1 and NRP-2 expressed on alveolar macrophages in lung tissue adjacent to cancer (squamous cell carcinoma), inflamed lung (interstitial pneumonia) and physiologically normal lung (remote to the cancer nest). NRP-1, neuropilin 1; NRP-2, neuropilin 2.
Table 3.
Comparison of neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) expression on alveolar macrophages in lung cancer adjacent to the cancer margin, lung inflammation and lung tissue remote to the cancer nest (physiologically normal lung).
Fig 7.
Double immunostaining (NRPs vs CD68, CD163, HO-1 and DC-SIGN) and triple immunohistochemistry (CD68, CD163 and NRP-1) of lung tissue adjacent to the cancer.
(A) Double IF showed expression of CD68, CD163 and HO-1 (Rhodamine, anti-mouse, red color) on NRP-1+(Fluorescein, anti-rabbit, green color) alveolar macrophages. White arrows showed double positive cells. Single IHC after single IF showed NRP-2+ (Rhodamine, anti-mouse, red color) alveolar macrophages also express CD68, CD163 and HO-1 (LSAB, anti-mouse, brown color). (B) (i) Double IHC showed NRP-1(Red) and DC-SIGN (blue) positive alveolar macrophages. (ii) Double IF showed the co-expression of NRP-2 (Rhodamine, anti-mouse, red color) and DC-SIGN (Fluorescein, anti-rabbit, green color) on AMs. (iii) Triple IHC of CD68 (brown), CD163 (light red) and NRP-1 (light blue) showed triple-positive cells (CD68+CD163+NRP-1+; indicated by black arrow) and double-positive cells (CD68+NRP-1+, CD68+CD163+ and CD163+NRP-1+). NRP-1, neuropilin 1; NRP-2, neuropilin 2; DC-SIGN, dendritic cell-specific ICAM-3-grabbing nonintegrin.
Table 4.
Comparison of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) expression on alveolar macrophages in lung tissue adjacent to the cancer margin and lung tissue remote to the cancer nest.