Limits...
Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Lazdins JK, Grell M, Walker MR, Woods-Cook K, Scheurich P, Pfizenmaier K - J. Exp. Med. (1997)

Bottom Line: In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production.Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells.These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

View Article: PubMed Central - PubMed

Affiliation: Division Pharma, Ciba, Basel, Switzerland.

ABSTRACT
Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Show MeSH

Related in: MedlinePlus

Effect of 3T3mTNF  cells on in vitro HIV-1-infected  and noninfected human lymphocytes. PBLs prepared from three  different healthy blood donors  were in vitro activated with  PHA (0.25 μg/ml) for 2 d, left  noninfected or were infected  with HIV-1/LAV. 3 d later, activated lymphocytes were plated  on a near confluent monolayer of  mTNF expressing 3T3mTNF  transfectants (mTNF) or 3T3  control cells without (control) or  with sTNF (10 ng/ml) (sTNF)  (six replicate cultures). At the  onset of cocultures, the RT activity of HIV-infected cells was  2219 ± 247 (donor 1), 585 ± 88  (donor 2) and 332 ± 62 (donor 3)  cpm/μl. (A) 16 h after coculture,  replicate cultures were pooled,  the YOPRO negative number  of cells analysed and the average  number of viable cells per well  was determined. Black bars indicate noninfected cells and grey bars indicate infected cells. (B) HIV-1 production (cell-free supernatant RT activity) of  the infected lymphocytes from the three donors upon separation from the 3T3 coculture after 16 h and replating in wells without 3T3 for an additional  2 d. The carry over of 3T3 cells was minimal as assessed by the absence of adherent cells after the 2-d culture. Each group represents the mean ± SD  from six replicate cultures.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196100&req=5

Figure 7: Effect of 3T3mTNF cells on in vitro HIV-1-infected and noninfected human lymphocytes. PBLs prepared from three different healthy blood donors were in vitro activated with PHA (0.25 μg/ml) for 2 d, left noninfected or were infected with HIV-1/LAV. 3 d later, activated lymphocytes were plated on a near confluent monolayer of mTNF expressing 3T3mTNF transfectants (mTNF) or 3T3 control cells without (control) or with sTNF (10 ng/ml) (sTNF) (six replicate cultures). At the onset of cocultures, the RT activity of HIV-infected cells was 2219 ± 247 (donor 1), 585 ± 88 (donor 2) and 332 ± 62 (donor 3) cpm/μl. (A) 16 h after coculture, replicate cultures were pooled, the YOPRO negative number of cells analysed and the average number of viable cells per well was determined. Black bars indicate noninfected cells and grey bars indicate infected cells. (B) HIV-1 production (cell-free supernatant RT activity) of the infected lymphocytes from the three donors upon separation from the 3T3 coculture after 16 h and replating in wells without 3T3 for an additional 2 d. The carry over of 3T3 cells was minimal as assessed by the absence of adherent cells after the 2-d culture. Each group represents the mean ± SD from six replicate cultures.

Mentions: To confirm the above described observations in a cellular system that reflects more closely the in vivo situation, we studied the role of mTNF in induction of apoptosis and virus production in HIV-infected, PHA-activated peripheral blood lymphocytes. The effect of mTNF was compared to that of sTNF in HIV-infected and noninfected PBLs. Fig. 7 shows results obtained in independent experiments with three different blood donors. 16–18 h after onset of cocultures, a significantly higher reduction in viable cell numbers was observed in all cultures containing mTNF expressing 3T3 cells as compared to cultures with control 3T3 cells plus high concentrations of sTNF. Although mTNF was found to induce apoptosis in both, the infected and the noninfected, PHA-activated T cells, in two out of the three donors examined here (numbers 2 and 3), an apparent preferential kill of the HIV-infected population (number of viable cells 13 and 30% of control) as compared to noninfected cultures (number of viable cells 50 and 58% of control) was observed (Fig. 7 A). More revealing, the T cells surviving a 16 h 3T3mTNF coculture were significantly impaired in their virus production capacity, as revealed upon transfer and reculture in the absence of mTNF expressing cells (Fig. 7 B), suggesting that the number of infected, virus producing cells has been reduced. In contrast, upon replating HIV-infected T cells pretreated for 16 h with sTNF RT activity was similar to control cultures. These data indicate a higher sensitivity of HIV-infected, primary lymphocytes towards apoptosis induced by mTNF.


Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Lazdins JK, Grell M, Walker MR, Woods-Cook K, Scheurich P, Pfizenmaier K - J. Exp. Med. (1997)

Effect of 3T3mTNF  cells on in vitro HIV-1-infected  and noninfected human lymphocytes. PBLs prepared from three  different healthy blood donors  were in vitro activated with  PHA (0.25 μg/ml) for 2 d, left  noninfected or were infected  with HIV-1/LAV. 3 d later, activated lymphocytes were plated  on a near confluent monolayer of  mTNF expressing 3T3mTNF  transfectants (mTNF) or 3T3  control cells without (control) or  with sTNF (10 ng/ml) (sTNF)  (six replicate cultures). At the  onset of cocultures, the RT activity of HIV-infected cells was  2219 ± 247 (donor 1), 585 ± 88  (donor 2) and 332 ± 62 (donor 3)  cpm/μl. (A) 16 h after coculture,  replicate cultures were pooled,  the YOPRO negative number  of cells analysed and the average  number of viable cells per well  was determined. Black bars indicate noninfected cells and grey bars indicate infected cells. (B) HIV-1 production (cell-free supernatant RT activity) of  the infected lymphocytes from the three donors upon separation from the 3T3 coculture after 16 h and replating in wells without 3T3 for an additional  2 d. The carry over of 3T3 cells was minimal as assessed by the absence of adherent cells after the 2-d culture. Each group represents the mean ± SD  from six replicate cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196100&req=5

Figure 7: Effect of 3T3mTNF cells on in vitro HIV-1-infected and noninfected human lymphocytes. PBLs prepared from three different healthy blood donors were in vitro activated with PHA (0.25 μg/ml) for 2 d, left noninfected or were infected with HIV-1/LAV. 3 d later, activated lymphocytes were plated on a near confluent monolayer of mTNF expressing 3T3mTNF transfectants (mTNF) or 3T3 control cells without (control) or with sTNF (10 ng/ml) (sTNF) (six replicate cultures). At the onset of cocultures, the RT activity of HIV-infected cells was 2219 ± 247 (donor 1), 585 ± 88 (donor 2) and 332 ± 62 (donor 3) cpm/μl. (A) 16 h after coculture, replicate cultures were pooled, the YOPRO negative number of cells analysed and the average number of viable cells per well was determined. Black bars indicate noninfected cells and grey bars indicate infected cells. (B) HIV-1 production (cell-free supernatant RT activity) of the infected lymphocytes from the three donors upon separation from the 3T3 coculture after 16 h and replating in wells without 3T3 for an additional 2 d. The carry over of 3T3 cells was minimal as assessed by the absence of adherent cells after the 2-d culture. Each group represents the mean ± SD from six replicate cultures.
Mentions: To confirm the above described observations in a cellular system that reflects more closely the in vivo situation, we studied the role of mTNF in induction of apoptosis and virus production in HIV-infected, PHA-activated peripheral blood lymphocytes. The effect of mTNF was compared to that of sTNF in HIV-infected and noninfected PBLs. Fig. 7 shows results obtained in independent experiments with three different blood donors. 16–18 h after onset of cocultures, a significantly higher reduction in viable cell numbers was observed in all cultures containing mTNF expressing 3T3 cells as compared to cultures with control 3T3 cells plus high concentrations of sTNF. Although mTNF was found to induce apoptosis in both, the infected and the noninfected, PHA-activated T cells, in two out of the three donors examined here (numbers 2 and 3), an apparent preferential kill of the HIV-infected population (number of viable cells 13 and 30% of control) as compared to noninfected cultures (number of viable cells 50 and 58% of control) was observed (Fig. 7 A). More revealing, the T cells surviving a 16 h 3T3mTNF coculture were significantly impaired in their virus production capacity, as revealed upon transfer and reculture in the absence of mTNF expressing cells (Fig. 7 B), suggesting that the number of infected, virus producing cells has been reduced. In contrast, upon replating HIV-infected T cells pretreated for 16 h with sTNF RT activity was similar to control cultures. These data indicate a higher sensitivity of HIV-infected, primary lymphocytes towards apoptosis induced by mTNF.

Bottom Line: In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production.Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells.These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

View Article: PubMed Central - PubMed

Affiliation: Division Pharma, Ciba, Basel, Switzerland.

ABSTRACT
Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Show MeSH
Related in: MedlinePlus