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Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Janatpour MJ, Hudak S, Sathe M, Sedgwick JD, McEvoy LM - J. Exp. Med. (2001)

Bottom Line: Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG.HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding.Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

View Article: PubMed Central - PubMed

Affiliation: DNAX Research Institute, Inc., Palo Alto, CA 94304, USA.

ABSTRACT
Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

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Real-time quantitative PCR (TaqMan™) analyses of chemokine expression in lymph nodes. Total RNA was isolated from pools of lymph nodes draining inflamed footpads (black bars) and pools of lymph nodes draining the contralateral footpads that were not inflamed (Normal, white bars). Real-time quantitative PCR was performed on 50 ng of reverse-transcribed cDNA using primers that specifically recognize a panel of chemokines. (A) mRNA levels for the subset of chemokines known to chemoattract monocytes in vitro and for MIG are shown. Data is expressed as fg per 50 ng cDNA. With the exception of RANTES, there was a relative increase in all the chemokines known to chemoattract monocytes, upon inflammation. P value < 0.005. Additionally, there was a greater than ninefold increase in the mRNA levels of MIG. (B) mRNA levels for a subset of chemokines in normal (not inflamed) and inflamed lymph nodes from TNF  mice are shown.
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fig1: Real-time quantitative PCR (TaqMan™) analyses of chemokine expression in lymph nodes. Total RNA was isolated from pools of lymph nodes draining inflamed footpads (black bars) and pools of lymph nodes draining the contralateral footpads that were not inflamed (Normal, white bars). Real-time quantitative PCR was performed on 50 ng of reverse-transcribed cDNA using primers that specifically recognize a panel of chemokines. (A) mRNA levels for the subset of chemokines known to chemoattract monocytes in vitro and for MIG are shown. Data is expressed as fg per 50 ng cDNA. With the exception of RANTES, there was a relative increase in all the chemokines known to chemoattract monocytes, upon inflammation. P value < 0.005. Additionally, there was a greater than ninefold increase in the mRNA levels of MIG. (B) mRNA levels for a subset of chemokines in normal (not inflamed) and inflamed lymph nodes from TNF mice are shown.

Mentions: Total RNA was extracted using RNA Stat 60 according to the manufacturer's instructions (CS110; Tel-Test). Total RNA was treated with DNase I (776-785; Roche Laboratories) to eliminate possible genomic DNA contamination. Total RNA was then reverse transcribed with oligo(dT)14–18 (18418; Life Technologies), random hexamers (C1181; Promega), dNTPs (27-2035; Amersham Pharmacia Biotech), and Superscript II (18064-014; Life Technologies), according to the manufacturer's instructions. 50 ng of cDNA was amplified using 12.5 μl of TaqMan™ universal master mix (PerkinElmer), 0.2 μM of gene-specific TaqMan™ probe, and 0.9 μM of gene-specific forward and reverse primers in a final volume of 25 μl. Primers and probes specific to each chemokine tested (see Fig. 1) were obtained from PerkinElmer. Ubiquitin mRNA levels were used to normalize between samples. Gene-specific PCR products were measured at each cycle, through 40 cycles, by means of a GeneAmp 5700 Sequence Detection System (PerkinElmer). mRNA levels were quantitated by comparing experimental levels to standard curves generated using serial dilutions of plasmids containing the chemokine gene being evaluated.


Tumor necrosis factor-dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment.

Janatpour MJ, Hudak S, Sathe M, Sedgwick JD, McEvoy LM - J. Exp. Med. (2001)

Real-time quantitative PCR (TaqMan™) analyses of chemokine expression in lymph nodes. Total RNA was isolated from pools of lymph nodes draining inflamed footpads (black bars) and pools of lymph nodes draining the contralateral footpads that were not inflamed (Normal, white bars). Real-time quantitative PCR was performed on 50 ng of reverse-transcribed cDNA using primers that specifically recognize a panel of chemokines. (A) mRNA levels for the subset of chemokines known to chemoattract monocytes in vitro and for MIG are shown. Data is expressed as fg per 50 ng cDNA. With the exception of RANTES, there was a relative increase in all the chemokines known to chemoattract monocytes, upon inflammation. P value < 0.005. Additionally, there was a greater than ninefold increase in the mRNA levels of MIG. (B) mRNA levels for a subset of chemokines in normal (not inflamed) and inflamed lymph nodes from TNF  mice are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195975&req=5

fig1: Real-time quantitative PCR (TaqMan™) analyses of chemokine expression in lymph nodes. Total RNA was isolated from pools of lymph nodes draining inflamed footpads (black bars) and pools of lymph nodes draining the contralateral footpads that were not inflamed (Normal, white bars). Real-time quantitative PCR was performed on 50 ng of reverse-transcribed cDNA using primers that specifically recognize a panel of chemokines. (A) mRNA levels for the subset of chemokines known to chemoattract monocytes in vitro and for MIG are shown. Data is expressed as fg per 50 ng cDNA. With the exception of RANTES, there was a relative increase in all the chemokines known to chemoattract monocytes, upon inflammation. P value < 0.005. Additionally, there was a greater than ninefold increase in the mRNA levels of MIG. (B) mRNA levels for a subset of chemokines in normal (not inflamed) and inflamed lymph nodes from TNF mice are shown.
Mentions: Total RNA was extracted using RNA Stat 60 according to the manufacturer's instructions (CS110; Tel-Test). Total RNA was treated with DNase I (776-785; Roche Laboratories) to eliminate possible genomic DNA contamination. Total RNA was then reverse transcribed with oligo(dT)14–18 (18418; Life Technologies), random hexamers (C1181; Promega), dNTPs (27-2035; Amersham Pharmacia Biotech), and Superscript II (18064-014; Life Technologies), according to the manufacturer's instructions. 50 ng of cDNA was amplified using 12.5 μl of TaqMan™ universal master mix (PerkinElmer), 0.2 μM of gene-specific TaqMan™ probe, and 0.9 μM of gene-specific forward and reverse primers in a final volume of 25 μl. Primers and probes specific to each chemokine tested (see Fig. 1) were obtained from PerkinElmer. Ubiquitin mRNA levels were used to normalize between samples. Gene-specific PCR products were measured at each cycle, through 40 cycles, by means of a GeneAmp 5700 Sequence Detection System (PerkinElmer). mRNA levels were quantitated by comparing experimental levels to standard curves generated using serial dilutions of plasmids containing the chemokine gene being evaluated.

Bottom Line: Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG.HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding.Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

View Article: PubMed Central - PubMed

Affiliation: DNAX Research Institute, Inc., Palo Alto, CA 94304, USA.

ABSTRACT
Monocytes recruited from the blood are key contributors to the nature of an immune response. While monocyte recruitment in a subset of immunopathologies has been well studied and largely attributed to the chemokine monocyte chemoattractant protein (MCP)-1, mechanisms mediating such recruitment to other sites of inflammation remain elusive. Here, we showed that localized inflammation resulted in an increased binding of monocytes to perifollicular high endothelial venules (HEVs) of lymph nodes draining a local inflammatory site. Quantitative PCR analyses revealed the upregulation of many chemokines in the inflamed lymph node, including MCP-1 and MIG. HEVs did not express detectable levels of MCP-1; however, a subset of HEVs in inflamed lymph nodes in wild-type (but not tumor necrosis factor [TNF] mice) expressed MIG and this subset of HEVs preferentially supported monocyte binding. Expression of CXCR3, the receptor for MIG, was detected on a small subset of peripheral blood monocytes and on a significant percentage of recruited monocytes. Most importantly, in both ex vivo and in vivo assays, neutralizing anti-MIG antibodies blocked monocyte binding to inflamed lymph node HEVs. Together, these results suggest that the lymph node microenvironment can dictate the nature of molecules expressed on HEV subsets in a TNF-dependent fashion and that inflammation-induced MIG expression by HEVs can mediate monocyte recruitment.

Show MeSH
Related in: MedlinePlus