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DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-lymphocytes.

de Witte L, de Vries RD, van der Vlist M, Yüksel S, Litjens M, de Swart RL, Geijtenbeek TB - PLoS Pathog. (2008)

Bottom Line: Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract.DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro.However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Measles virus (MV) is among the most infectious viruses that affect humans and is transmitted via the respiratory route. In macaques, MV primarily infects lymphocytes and dendritic cells (DCs). Little is known about the initial target cell for MV infection. Since DCs bridge the peripheral mucosal tissues with lymphoid tissues, we hypothesize that DCs are the initial target cells that capture MV in the respiratory tract and transport the virus to the lymphoid tissues where MV is transmitted to lymphocytes. Recently, we have demonstrated that the C-type lectin DC-SIGN interacts with MV and enhances infection of DCs in cis. Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract. DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro. However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes. We distinguished two different transmission routes that were either dependent or independent on direct DC infection. DC-SIGN and CD150 are both involved in direct DC infection and subsequent transmission of de novo synthesized virus. However, DC-SIGN, but not CD150, mediates trans-infection of MV to T-lymphocytes independent of DC infection. Together these data suggest a prominent role for DCs during the initiation, dissemination, and clearance of MV infection.

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Dendritic cells mediate viral transmission of measles virus independently of de novo virus production.(A–D) DCs (5×104 cells) were incubated with MV-IC323-EGFP (5×104TCID50, unless depicted otherwise) and after 2 hours the cells were extensively washed. T-lymphocytes (2×105 cells) were added and if indicated the fusion inhibitory peptide (FIP) was added after 2 hours. After 72 hours the infection was analyzed by (A) fluorescence microscopy and (B–D) flow cytometry. (A) The cultures are depicted as an overlay of EGFP and brightfield. (B,C) The cells were harvested, washed and stained for CD3 and DC-SIGN. EGFP expression was measured by flow cytometry. (B) The percentage gated is depicted in the regions or quadrants. (C) Transmission of different concentrations of MV IC323-EGFP is analyzed. (D) The absolute numbers of EGFP+ DCs and T-lymphocytes in the analyzed samples were calculated and depicted. Error bars represent the standard deviation of triplicates. A representative donor out of seven is shown.
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ppat-1000049-g004: Dendritic cells mediate viral transmission of measles virus independently of de novo virus production.(A–D) DCs (5×104 cells) were incubated with MV-IC323-EGFP (5×104TCID50, unless depicted otherwise) and after 2 hours the cells were extensively washed. T-lymphocytes (2×105 cells) were added and if indicated the fusion inhibitory peptide (FIP) was added after 2 hours. After 72 hours the infection was analyzed by (A) fluorescence microscopy and (B–D) flow cytometry. (A) The cultures are depicted as an overlay of EGFP and brightfield. (B,C) The cells were harvested, washed and stained for CD3 and DC-SIGN. EGFP expression was measured by flow cytometry. (B) The percentage gated is depicted in the regions or quadrants. (C) Transmission of different concentrations of MV IC323-EGFP is analyzed. (D) The absolute numbers of EGFP+ DCs and T-lymphocytes in the analyzed samples were calculated and depicted. Error bars represent the standard deviation of triplicates. A representative donor out of seven is shown.

Mentions: To investigate whether DCs transmit MV to their target cells, DCs were incubated with MV-IC323-EGFP for two hours, washed extensively to remove unbound virus and subsequently co-cultured with activated T-lymphocytes. In DC cultures without T-lymphocytes, low percentages of MV-infected DCs were detected, whereas in the DC-T lymphocyte co-cultures large clusters of MV-infected cells and syncytia were observed (Figure 4A upper panels). These data strongly suggest that DCs capture MV and transmit the virus to T-lymphocytes independently of de novo synthesis of virus by infected DCs, since only a few infected DCs were observed (Figure 4A). This process is referred to as trans-infection. However, HIV-1 studies have shown that DCs can also mediate transmission of de novo synthesized HIV-1 [10]. DCs in the respiratory tract in situ express high levels of DC-SIGN and no or low amounts of CD150 (Figure 1), suggesting that DCs are not productively infected by MV. Therefore, we investigated whether DCs mediate trans-infection. As demonstrated, DCs transmit MV efficiently to T-lymphocytes in a co-culture (Figure 4A) but de novo synthesis in DCs cannot be excluded, since both DCs and T-lymphocytes are infected in the DC-T lymphocyte co-culture (Figure 4B). To exclude de novo synthesis of virus in DCs, we used the fusion inhibitor peptide (FIP, 200µM) [23], which was added to the co-cultures 2 hours after addition of the T-lymphocytes to MV-infected DCs. We observed large clusters of EGFP+ cells in the presence of FIP (Figure 4A). FIP prevents fusion of MV with cell membranes and of membranes of MV-infected cells with those of neighbouring cells. Therefore FIP blocks infection and syncytium formation [24]. Thus, T-lymphocytes expressing EGFP must have been infected during the 2 hours co-cultivation with MV-infected DCs before FIP was added (Figure 4B). This is a time frame that excludes de novo synthesis of MV by the DCs. In contrast to the condition without FIP, no syncytium formation was observed in DCs and T-lymphocytes cultured in the presence of FIP, confirming that FIP indeed prevented fusion. Moreover, incubation of DCs with different concentrations of MV demonstrated that T-lymphocytes were the major MV-infected cell population in the DC-T lymphocyte co-culture (Figure 4C), demonstrating that DCs mediate trans-infection. To determine the efficiency of the trans-infection, the absolute number of infected cells was calculated (Figure 4D). A 6-fold higher number of T-lymphocytes compared to DCs were infected in the co-cultures. This demonstrates that trans-infection of T-lymphocytes by DC-bound MV is more efficient than cis-infection of DCs. Thus, DCs efficiently mediate transmission of MV to T-lymphocytes, and this process primarily occurs independently of de novo synthesis of MV.


DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-lymphocytes.

de Witte L, de Vries RD, van der Vlist M, Yüksel S, Litjens M, de Swart RL, Geijtenbeek TB - PLoS Pathog. (2008)

Dendritic cells mediate viral transmission of measles virus independently of de novo virus production.(A–D) DCs (5×104 cells) were incubated with MV-IC323-EGFP (5×104TCID50, unless depicted otherwise) and after 2 hours the cells were extensively washed. T-lymphocytes (2×105 cells) were added and if indicated the fusion inhibitory peptide (FIP) was added after 2 hours. After 72 hours the infection was analyzed by (A) fluorescence microscopy and (B–D) flow cytometry. (A) The cultures are depicted as an overlay of EGFP and brightfield. (B,C) The cells were harvested, washed and stained for CD3 and DC-SIGN. EGFP expression was measured by flow cytometry. (B) The percentage gated is depicted in the regions or quadrants. (C) Transmission of different concentrations of MV IC323-EGFP is analyzed. (D) The absolute numbers of EGFP+ DCs and T-lymphocytes in the analyzed samples were calculated and depicted. Error bars represent the standard deviation of triplicates. A representative donor out of seven is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2277461&req=5

ppat-1000049-g004: Dendritic cells mediate viral transmission of measles virus independently of de novo virus production.(A–D) DCs (5×104 cells) were incubated with MV-IC323-EGFP (5×104TCID50, unless depicted otherwise) and after 2 hours the cells were extensively washed. T-lymphocytes (2×105 cells) were added and if indicated the fusion inhibitory peptide (FIP) was added after 2 hours. After 72 hours the infection was analyzed by (A) fluorescence microscopy and (B–D) flow cytometry. (A) The cultures are depicted as an overlay of EGFP and brightfield. (B,C) The cells were harvested, washed and stained for CD3 and DC-SIGN. EGFP expression was measured by flow cytometry. (B) The percentage gated is depicted in the regions or quadrants. (C) Transmission of different concentrations of MV IC323-EGFP is analyzed. (D) The absolute numbers of EGFP+ DCs and T-lymphocytes in the analyzed samples were calculated and depicted. Error bars represent the standard deviation of triplicates. A representative donor out of seven is shown.
Mentions: To investigate whether DCs transmit MV to their target cells, DCs were incubated with MV-IC323-EGFP for two hours, washed extensively to remove unbound virus and subsequently co-cultured with activated T-lymphocytes. In DC cultures without T-lymphocytes, low percentages of MV-infected DCs were detected, whereas in the DC-T lymphocyte co-cultures large clusters of MV-infected cells and syncytia were observed (Figure 4A upper panels). These data strongly suggest that DCs capture MV and transmit the virus to T-lymphocytes independently of de novo synthesis of virus by infected DCs, since only a few infected DCs were observed (Figure 4A). This process is referred to as trans-infection. However, HIV-1 studies have shown that DCs can also mediate transmission of de novo synthesized HIV-1 [10]. DCs in the respiratory tract in situ express high levels of DC-SIGN and no or low amounts of CD150 (Figure 1), suggesting that DCs are not productively infected by MV. Therefore, we investigated whether DCs mediate trans-infection. As demonstrated, DCs transmit MV efficiently to T-lymphocytes in a co-culture (Figure 4A) but de novo synthesis in DCs cannot be excluded, since both DCs and T-lymphocytes are infected in the DC-T lymphocyte co-culture (Figure 4B). To exclude de novo synthesis of virus in DCs, we used the fusion inhibitor peptide (FIP, 200µM) [23], which was added to the co-cultures 2 hours after addition of the T-lymphocytes to MV-infected DCs. We observed large clusters of EGFP+ cells in the presence of FIP (Figure 4A). FIP prevents fusion of MV with cell membranes and of membranes of MV-infected cells with those of neighbouring cells. Therefore FIP blocks infection and syncytium formation [24]. Thus, T-lymphocytes expressing EGFP must have been infected during the 2 hours co-cultivation with MV-infected DCs before FIP was added (Figure 4B). This is a time frame that excludes de novo synthesis of MV by the DCs. In contrast to the condition without FIP, no syncytium formation was observed in DCs and T-lymphocytes cultured in the presence of FIP, confirming that FIP indeed prevented fusion. Moreover, incubation of DCs with different concentrations of MV demonstrated that T-lymphocytes were the major MV-infected cell population in the DC-T lymphocyte co-culture (Figure 4C), demonstrating that DCs mediate trans-infection. To determine the efficiency of the trans-infection, the absolute number of infected cells was calculated (Figure 4D). A 6-fold higher number of T-lymphocytes compared to DCs were infected in the co-cultures. This demonstrates that trans-infection of T-lymphocytes by DC-bound MV is more efficient than cis-infection of DCs. Thus, DCs efficiently mediate transmission of MV to T-lymphocytes, and this process primarily occurs independently of de novo synthesis of MV.

Bottom Line: Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract.DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro.However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.

ABSTRACT
Measles virus (MV) is among the most infectious viruses that affect humans and is transmitted via the respiratory route. In macaques, MV primarily infects lymphocytes and dendritic cells (DCs). Little is known about the initial target cell for MV infection. Since DCs bridge the peripheral mucosal tissues with lymphoid tissues, we hypothesize that DCs are the initial target cells that capture MV in the respiratory tract and transport the virus to the lymphoid tissues where MV is transmitted to lymphocytes. Recently, we have demonstrated that the C-type lectin DC-SIGN interacts with MV and enhances infection of DCs in cis. Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract. DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro. However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes. We distinguished two different transmission routes that were either dependent or independent on direct DC infection. DC-SIGN and CD150 are both involved in direct DC infection and subsequent transmission of de novo synthesized virus. However, DC-SIGN, but not CD150, mediates trans-infection of MV to T-lymphocytes independent of DC infection. Together these data suggest a prominent role for DCs during the initiation, dissemination, and clearance of MV infection.

Show MeSH
Related in: MedlinePlus