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C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Verani A, Scarlatti G, Comar M, Tresoldi E, Polo S, Giacca M, Lusso P, Siccardi AG, Vercelli D - J. Exp. Med. (1997)

Bottom Line: A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS.Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages.Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT
Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

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MDM express CC–CKR-5 mRNA. Total RNA was extracted from untreated MDM. RNA samples were treated with DNase I  to remove traces of contaminating DNA and reverse transcribed using  random hexameric primers. The cDNA products were mixed to scalar  amounts of a synthetic competitor DNA fragment containing primer recognition sites for both β-actin and CC–CKR-5 amplification, and amplified with the respective primer pairs. (A) Schematic representation of the  competitor DNA fragment used for the quantification of CC–CKR-5  and β-actin cDNA. The fragment contains a core sequence derived from  the human β-actin cDNA, carrying a 20-bp insertion in the middle (closed  box). Amplification with the β-actin-specific primer set BA1–BA4 detects  a 226-bp product on human cDNA, and a 246-bp product from the  competitor DNA. To this core sequence, the primer recognition sites for  human CC–CKR-5 amplification were added at the two ends (indicated  by gray boxes) by reamplification with composite primers corresponding  to the CKR-9+BA1 sequence at one end and CKR-10+BA4 at the  other end. Amplification with CKR-9 and CKR-10 generates a 288-bp  fragment from the competitor template and a 368-bp fragment from the  CC–CKR-5 cDNA. (B) Competitive PCR for the quantification of CC– CKR-5 and β-actin mRNAs. cDNA samples from untreated MDM  were mixed with tenfold dilution of the competitor DNA fragment as indicated, and amplified with primer sets CKR-9/CKR-10 and BA1/BA4  for CC–CKR-5 and β-actin mRNA quantification. Amplification products were resolved by polyacrylamide gel electrophoresis, stained with  ethidium bromide, and quantified by densitometric scanning. According  to the principles of competitive PCR, quantification of the target molecules in the samples was obtained by estimation of the ratio between the  amplification products, as reported at the bottom of each gel. Furthermore,  since the same competitor DNA fragment acts as a competitor for quantification of both CC–CKR-5 and β-actin, standardization for mRNA input is  obtained by estimating the ratio between the two measurements, as indicated at the bottom of the figure. M, molecular weight markers.
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Figure 6: MDM express CC–CKR-5 mRNA. Total RNA was extracted from untreated MDM. RNA samples were treated with DNase I to remove traces of contaminating DNA and reverse transcribed using random hexameric primers. The cDNA products were mixed to scalar amounts of a synthetic competitor DNA fragment containing primer recognition sites for both β-actin and CC–CKR-5 amplification, and amplified with the respective primer pairs. (A) Schematic representation of the competitor DNA fragment used for the quantification of CC–CKR-5 and β-actin cDNA. The fragment contains a core sequence derived from the human β-actin cDNA, carrying a 20-bp insertion in the middle (closed box). Amplification with the β-actin-specific primer set BA1–BA4 detects a 226-bp product on human cDNA, and a 246-bp product from the competitor DNA. To this core sequence, the primer recognition sites for human CC–CKR-5 amplification were added at the two ends (indicated by gray boxes) by reamplification with composite primers corresponding to the CKR-9+BA1 sequence at one end and CKR-10+BA4 at the other end. Amplification with CKR-9 and CKR-10 generates a 288-bp fragment from the competitor template and a 368-bp fragment from the CC–CKR-5 cDNA. (B) Competitive PCR for the quantification of CC– CKR-5 and β-actin mRNAs. cDNA samples from untreated MDM were mixed with tenfold dilution of the competitor DNA fragment as indicated, and amplified with primer sets CKR-9/CKR-10 and BA1/BA4 for CC–CKR-5 and β-actin mRNA quantification. Amplification products were resolved by polyacrylamide gel electrophoresis, stained with ethidium bromide, and quantified by densitometric scanning. According to the principles of competitive PCR, quantification of the target molecules in the samples was obtained by estimation of the ratio between the amplification products, as reported at the bottom of each gel. Furthermore, since the same competitor DNA fragment acts as a competitor for quantification of both CC–CKR-5 and β-actin, standardization for mRNA input is obtained by estimating the ratio between the two measurements, as indicated at the bottom of the figure. M, molecular weight markers.

Mentions: This procedure was described in detail elsewhere (20). In brief, total RNA was extracted according to the guanidine thiocyanate procedure (21), and treated with RNase-free DNase I (Boehringer, Mannheim, Germany) to remove traces of contaminating DNA. First-strand cDNA synthesis was obtained by priming with random hexamers and reverse transcription in 20 μl of RT mix containing 75 mM KCl, 50 mM Tris–HCl (pH 8.3), 3 mM MgCl2, 0.4 mM each dNTP (Pharmacia), 400 U Moloney murine leukemia virus (MMLV)–RT (Promega, Madison, WI), 20 U RNasin (Promega). RNA was preheated at 65°C for 5 min and incubated with the reaction mix at 37°C. After 1 h, the reaction was stopped by incubation at 95°C for 5 min and samples were cooled on ice. Amplification of CC–CKR-5 cDNA was performed using primers CKR-9 (5′-CATCATCCTCCTGACAATCG) and CKR-10 (5′-ATGGTGAAGATAAGCCTCACAG). Quantification of CC–CKR-5 mRNA levels in MDM was carried out by a competitive PCR procedure using a competitor DNA fragment carrying the primer recognition sites for β-actin (BA1 and BA4 [22]) and for CKR-5 (primers CKR-9 and CKR-10). β-actin is used as a standard to monitor the efficiency of total DNA extraction. A schematic representation of this competitor is shown in Fig. 6 A and its construction is described in the legend to Fig. 6.


C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells.

Verani A, Scarlatti G, Comar M, Tresoldi E, Polo S, Giacca M, Lusso P, Siccardi AG, Vercelli D - J. Exp. Med. (1997)

MDM express CC–CKR-5 mRNA. Total RNA was extracted from untreated MDM. RNA samples were treated with DNase I  to remove traces of contaminating DNA and reverse transcribed using  random hexameric primers. The cDNA products were mixed to scalar  amounts of a synthetic competitor DNA fragment containing primer recognition sites for both β-actin and CC–CKR-5 amplification, and amplified with the respective primer pairs. (A) Schematic representation of the  competitor DNA fragment used for the quantification of CC–CKR-5  and β-actin cDNA. The fragment contains a core sequence derived from  the human β-actin cDNA, carrying a 20-bp insertion in the middle (closed  box). Amplification with the β-actin-specific primer set BA1–BA4 detects  a 226-bp product on human cDNA, and a 246-bp product from the  competitor DNA. To this core sequence, the primer recognition sites for  human CC–CKR-5 amplification were added at the two ends (indicated  by gray boxes) by reamplification with composite primers corresponding  to the CKR-9+BA1 sequence at one end and CKR-10+BA4 at the  other end. Amplification with CKR-9 and CKR-10 generates a 288-bp  fragment from the competitor template and a 368-bp fragment from the  CC–CKR-5 cDNA. (B) Competitive PCR for the quantification of CC– CKR-5 and β-actin mRNAs. cDNA samples from untreated MDM  were mixed with tenfold dilution of the competitor DNA fragment as indicated, and amplified with primer sets CKR-9/CKR-10 and BA1/BA4  for CC–CKR-5 and β-actin mRNA quantification. Amplification products were resolved by polyacrylamide gel electrophoresis, stained with  ethidium bromide, and quantified by densitometric scanning. According  to the principles of competitive PCR, quantification of the target molecules in the samples was obtained by estimation of the ratio between the  amplification products, as reported at the bottom of each gel. Furthermore,  since the same competitor DNA fragment acts as a competitor for quantification of both CC–CKR-5 and β-actin, standardization for mRNA input is  obtained by estimating the ratio between the two measurements, as indicated at the bottom of the figure. M, molecular weight markers.
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Figure 6: MDM express CC–CKR-5 mRNA. Total RNA was extracted from untreated MDM. RNA samples were treated with DNase I to remove traces of contaminating DNA and reverse transcribed using random hexameric primers. The cDNA products were mixed to scalar amounts of a synthetic competitor DNA fragment containing primer recognition sites for both β-actin and CC–CKR-5 amplification, and amplified with the respective primer pairs. (A) Schematic representation of the competitor DNA fragment used for the quantification of CC–CKR-5 and β-actin cDNA. The fragment contains a core sequence derived from the human β-actin cDNA, carrying a 20-bp insertion in the middle (closed box). Amplification with the β-actin-specific primer set BA1–BA4 detects a 226-bp product on human cDNA, and a 246-bp product from the competitor DNA. To this core sequence, the primer recognition sites for human CC–CKR-5 amplification were added at the two ends (indicated by gray boxes) by reamplification with composite primers corresponding to the CKR-9+BA1 sequence at one end and CKR-10+BA4 at the other end. Amplification with CKR-9 and CKR-10 generates a 288-bp fragment from the competitor template and a 368-bp fragment from the CC–CKR-5 cDNA. (B) Competitive PCR for the quantification of CC– CKR-5 and β-actin mRNAs. cDNA samples from untreated MDM were mixed with tenfold dilution of the competitor DNA fragment as indicated, and amplified with primer sets CKR-9/CKR-10 and BA1/BA4 for CC–CKR-5 and β-actin mRNA quantification. Amplification products were resolved by polyacrylamide gel electrophoresis, stained with ethidium bromide, and quantified by densitometric scanning. According to the principles of competitive PCR, quantification of the target molecules in the samples was obtained by estimation of the ratio between the amplification products, as reported at the bottom of each gel. Furthermore, since the same competitor DNA fragment acts as a competitor for quantification of both CC–CKR-5 and β-actin, standardization for mRNA input is obtained by estimating the ratio between the two measurements, as indicated at the bottom of the figure. M, molecular weight markers.
Mentions: This procedure was described in detail elsewhere (20). In brief, total RNA was extracted according to the guanidine thiocyanate procedure (21), and treated with RNase-free DNase I (Boehringer, Mannheim, Germany) to remove traces of contaminating DNA. First-strand cDNA synthesis was obtained by priming with random hexamers and reverse transcription in 20 μl of RT mix containing 75 mM KCl, 50 mM Tris–HCl (pH 8.3), 3 mM MgCl2, 0.4 mM each dNTP (Pharmacia), 400 U Moloney murine leukemia virus (MMLV)–RT (Promega, Madison, WI), 20 U RNasin (Promega). RNA was preheated at 65°C for 5 min and incubated with the reaction mix at 37°C. After 1 h, the reaction was stopped by incubation at 95°C for 5 min and samples were cooled on ice. Amplification of CC–CKR-5 cDNA was performed using primers CKR-9 (5′-CATCATCCTCCTGACAATCG) and CKR-10 (5′-ATGGTGAAGATAAGCCTCACAG). Quantification of CC–CKR-5 mRNA levels in MDM was carried out by a competitive PCR procedure using a competitor DNA fragment carrying the primer recognition sites for β-actin (BA1 and BA4 [22]) and for CKR-5 (primers CKR-9 and CKR-10). β-actin is used as a standard to monitor the efficiency of total DNA extraction. A schematic representation of this competitor is shown in Fig. 6 A and its construction is described in the legend to Fig. 6.

Bottom Line: A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS.Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages.Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT
Human immunodeficiency virus-1 (HIV-1) expression in monocyte-derived macrophages (MDM) infected in vitro is known to be inhibited by lipopolysaccharide (LPS). However, the mechanisms are incompletely understood. We show here that HIV-1 suppression is mediated by soluble factors released by MDM stimulated with physiologically significant concentrations of LPS. LPS-conditioned supernatants from MDM inhibited HIV-1 replication in both MDM and T cells. Depletion of C-C chemokines (RANTES, MIP-1 alpha, and MIP-1 beta) neutralized the ability of LPS-conditioned supernatants to inhibit HIV-1 replication in MDM. A combination of recombinant C-C chemokines blocked HIV-1 infection as effectively as LPS. Here, we report an inhibitory effect of C-C chemokines on HIV replication in primary macrophages. Our results raise the possibility that monocytes may play a dual role in HIV infection: while representing a reservoir for the virus, they may contribute to the containment of the infection by releasing factors that suppress HIV replication not only in monocytes but also in T lymphocytes.

Show MeSH
Related in: MedlinePlus