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Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

Quinones M, Ahuja SK, Melby PC, Pate L, Reddick RL, Ahuja SS - J. Exp. Med. (2000)

Bottom Line: However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated.These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen.Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

View Article: PubMed Central - PubMed

Affiliation: South Texas Veterans Health Care System, Audie L. Murphy Division, University of Texas Health Science Center at San Antonio, Texas 78229-3900, USA.

ABSTRACT
The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

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Rapid release of bioactive, preformed IL-12 p70 after 10 min of contact with L. donovani (Ld) promastigotes. (A) IL-12 p70 was detected in the supernatants of DCs in contact with L. donovani for 10 min but not in the supernatants of DCs stimulated for the same duration with LPS plus IFN-γ. Pretreating (pretreat) DCs with actinomycin (Act), brefeldin A (Bref), or monensin (Mon) had no effect on the amounts of IL-12 p70 released from DCs in contact with L. donovani. (B) Supernatants (Sup) from unstimulated DCs or DCs in contact with L. donovani (DC + Ld) was added to Ag-reactive splenocytes (Spl). A blocking IL-12 Ab but not isotype control Ab reduced the amounts of IFN-γ that is released after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes. The amounts of IFN-γ released from splenocytes after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes was similar to that released after addition of recombinant IL-12 p70 (300 pg/ml).
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Figure 3: Rapid release of bioactive, preformed IL-12 p70 after 10 min of contact with L. donovani (Ld) promastigotes. (A) IL-12 p70 was detected in the supernatants of DCs in contact with L. donovani for 10 min but not in the supernatants of DCs stimulated for the same duration with LPS plus IFN-γ. Pretreating (pretreat) DCs with actinomycin (Act), brefeldin A (Bref), or monensin (Mon) had no effect on the amounts of IL-12 p70 released from DCs in contact with L. donovani. (B) Supernatants (Sup) from unstimulated DCs or DCs in contact with L. donovani (DC + Ld) was added to Ag-reactive splenocytes (Spl). A blocking IL-12 Ab but not isotype control Ab reduced the amounts of IFN-γ that is released after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes. The amounts of IFN-γ released from splenocytes after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes was similar to that released after addition of recombinant IL-12 p70 (300 pg/ml).

Mentions: The IL-12 p70 produced by unstimulated DCs was below the detection limits of the ELISA; however, after as little as 10 min of contact with stationary phase (or purified metacyclic) L. donovani promastigotes, murine DCs released IL-12 p70 (Fig. 3 A). This release was independent of de novo IL-12 synthesis, as pretreatment of DCs with actinomycin D (inhibitor of transcription) or monensin or brefeldin A (inhibitors of Golgi transport) had no effect on the amounts of IL-12 p70 released from DCs (Fig. 3 A).


Preformed membrane-associated stores of interleukin (IL)-12 are a previously unrecognized source of bioactive IL-12 that is mobilized within minutes of contact with an intracellular parasite.

Quinones M, Ahuja SK, Melby PC, Pate L, Reddick RL, Ahuja SS - J. Exp. Med. (2000)

Rapid release of bioactive, preformed IL-12 p70 after 10 min of contact with L. donovani (Ld) promastigotes. (A) IL-12 p70 was detected in the supernatants of DCs in contact with L. donovani for 10 min but not in the supernatants of DCs stimulated for the same duration with LPS plus IFN-γ. Pretreating (pretreat) DCs with actinomycin (Act), brefeldin A (Bref), or monensin (Mon) had no effect on the amounts of IL-12 p70 released from DCs in contact with L. donovani. (B) Supernatants (Sup) from unstimulated DCs or DCs in contact with L. donovani (DC + Ld) was added to Ag-reactive splenocytes (Spl). A blocking IL-12 Ab but not isotype control Ab reduced the amounts of IFN-γ that is released after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes. The amounts of IFN-γ released from splenocytes after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes was similar to that released after addition of recombinant IL-12 p70 (300 pg/ml).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193237&req=5

Figure 3: Rapid release of bioactive, preformed IL-12 p70 after 10 min of contact with L. donovani (Ld) promastigotes. (A) IL-12 p70 was detected in the supernatants of DCs in contact with L. donovani for 10 min but not in the supernatants of DCs stimulated for the same duration with LPS plus IFN-γ. Pretreating (pretreat) DCs with actinomycin (Act), brefeldin A (Bref), or monensin (Mon) had no effect on the amounts of IL-12 p70 released from DCs in contact with L. donovani. (B) Supernatants (Sup) from unstimulated DCs or DCs in contact with L. donovani (DC + Ld) was added to Ag-reactive splenocytes (Spl). A blocking IL-12 Ab but not isotype control Ab reduced the amounts of IFN-γ that is released after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes. The amounts of IFN-γ released from splenocytes after addition of supernatants from DC–Leishmania cocultures to Ag-reactive splenocytes was similar to that released after addition of recombinant IL-12 p70 (300 pg/ml).
Mentions: The IL-12 p70 produced by unstimulated DCs was below the detection limits of the ELISA; however, after as little as 10 min of contact with stationary phase (or purified metacyclic) L. donovani promastigotes, murine DCs released IL-12 p70 (Fig. 3 A). This release was independent of de novo IL-12 synthesis, as pretreatment of DCs with actinomycin D (inhibitor of transcription) or monensin or brefeldin A (inhibitors of Golgi transport) had no effect on the amounts of IL-12 p70 released from DCs (Fig. 3 A).

Bottom Line: However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated.These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen.Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

View Article: PubMed Central - PubMed

Affiliation: South Texas Veterans Health Care System, Audie L. Murphy Division, University of Texas Health Science Center at San Antonio, Texas 78229-3900, USA.

ABSTRACT
The prevailing paradigm is that production of the interleukin (IL)-12 p70 heterodimer, a critical T helper cell type 1 (Th1)-inducing cytokine, depends on the induced transcription of the p40 subunit. Concordant with this paradigm, we found that dendritic cells (DCs) produced IL-12 p70 only after at least 2-4 h of stimulation with lipopolysaccharide plus interferon gamma. However, using several complementary experimental approaches, including electron and confocal microscopy, we now show that resting murine and human myeloid cells, including macrophages/DCs and DC-rich tissues, contain a novel source of bioactive IL-12 that is preformed and membrane associated. These preformed, membrane-associated IL-12 p70 stores are released within minutes after in vitro or in vivo contact with Leishmania donovani, an intracellular pathogen. Our findings highlight a novel source of bioactive IL-12 that is readily available for the rapid initiation of Th1 host responses to pathogens such as Leishmania species.

Show MeSH
Related in: MedlinePlus