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Nonselective actions of herbimycin A
kaleidoscope letters Problems with metabolic inhibitors: suppressing normal assumptions. Two labs suggest care must be taken when using even the most well-described reagents.
Why cyclosporin is an effective drug There has been ~, recent spate of articles in Immunology Today concerning the mechanism of action of cyclosporin A (CsA). I am responsible for one and there are two others 1-3. It is riA:y to write about CsA mechanisms because, invariably, before the article appears in print, there are new, surprising findings that require rethinking of any prior hypothesis. All three articles (and others published elsewhere) have focussed on mechanisms by which CsA inhibits the synthesis and secretion of interleukin 2 (IL-2) by T cells. I now sugges'c that the efficacy of CsA as a drug in preventing organ rejection in humans does not rest on that ability. I contend that, in fact, inhibition of IL-2 synthesis can be a deleterious side effect of CsA. It puts the patient in hospital periodically with opportunistic infections. I propose that the key to the success of CsA as a drug in preventing organ rejection, and in the treatment of psoriasis, can be found in ,,,,, recent papers <5 .t_. t,at were
Nonselective actions of herbimycin A We wish to call attention to an unsuspected action of herbimycin A, which is widely used as an inhibitor of tyrosine kinases to define the role of these enzymes in Fc- and T-cell receptor signalling processes 1,-'. Typically, the inhibition of production of inositol phosphates by this reagent is attributed to suppression of tyrosine phosphorylation of phospholipase C vl by a tyrosine kinase. As indicated below, this assumption may not be valid even when herbimycin A does block such phosphorylation. The actions of herbimycin A are apparent only after cells have
added as a note to the proof of my Immunology Today article. They were not available ~lhen the article was submitted. Bc,th papers reported on extracellular, soluble cyclophilins. The Faper by Q. Xu, M.C. Leiva, M.K. Fischkoff et al. s is of particular interest because it reported that 'the extracellular cyclophilin was chemotactic for eosinophils and neutrophils, and that its chemotactic activity was inhibited by CsA. They pointed out that the ubiquitous tissue distribution of cyclophilin (it is present in all cells, even erythrocytes) may reflect the ability of various tissues to respond to injury through the release of cyclophilin resulting in leukocyte infiltration and clearing of the injured cells. Eosinophil infiltration has been associated with both organ graft rejection and psoriasis 3 and CsA is effective in inhibiting the infiltration. Xu et al. suggest that CsA does so by modulating chemotaxis. They propose that this mechanism may be complementary to the other activities of CsA, including inhibition of IL-2 synthesis. I propose that this mechanism, rather than being complementary I • -! to mecnamsms mat involve inhi-
been exposed to it for several hours to allow for complete degradation of the tyrosine kinase 3. Following the protocols of previous wo¢k4, we exposed rat RBL2H3 cells, a tumor analog of rat mucosal mast cells, to 800 aM herbimycin A or vehicle for 14 hours in the presence of growth medium, myo-I3Hlinositol (4 gtCi ml-t) and dinitrophenol-specific lgE, before washing the cells and stimulating them with antigen {20 ng ml-l dinitrophenylated bovine serum albumin) for 15 rain. The Table illustrates a sample experiment in which the incorporation of the inositol into the inositol phospholipid fraction was reduced by >80% in herbimycin-treated ce!ls. The amounts of radiolabeled
Immunology Today
369
bition of IL-2 synthesis, is the prime basis of the success of CsA in prolonging organ graft survival in patients, and in the treatment of psoriasis. It would follow, then, that the search should ,ow be for agents, including CsA derivatives, that strongly inhibit chemotaxis but are less effective in inhibiting iL-2 synthesis by T cells. Bernard F. Erlanger Dept of Microbiology, Columbia University, New York, NY 10032, USA. Re[erences
1 Schreiber, S.L. and Crabtree, G.R. (1992} Immunol. Today 13, 136-142 2 Erlanger, B.F. (1992) Immunol. Today 13, 487-490 3 Wong, R.L., Winslow, C.M. and Cooper, K.D. (1993) Immunol. Today 14, 69-74 4 Sherry, B., Yarlett, N., Strupp, A. and Cerami, A. (1992) Proc. Natl Acad. Sci. USA 89, 3511-3515 5 Xu, Q., LeNa, M.C., Fischkoff, M.K., Handschumacher, R.E. and Lyttle, C.R. (1992)J. BioL Chem. 267, i 1968-i 15~71
inositol in herbimycin A-treated and untreated cells were similar, but the amounts of radiolabeled inositol phospholipids and phosphates were reduced significantly in the treated cells. The distribution of radiolabel among the inositol phospholipid pools (phosphatidylinositol:phosphatidylinositol 4phosphate:phosphatidylinositol 4,5-bisphosphate) in untreated and treated cells, however, was the same namely, 95:2:3 in unstimulated cclis and 90:5:5 in ztimulated cells as determined by thin layer chromatography4. Results such as this indicate that herbimycin A may impede incorporation of inositol into the phospholipid pools. An even more striking result was obtained with RBL-2H3 cells that
vol 14 No. 71993
kaleidoscope letters Treatment
[3H]inositol phosphates
[3H]myoinositol
[3H]inositoi phospholipids
dpm/0.3xl06 cells Untreated
Unstimulated Antigen
450+._27 5994+185
Herbim.-A
Unstimulated Antigen
125_+7 237+-26
had been transfected with the gene for the G protein-coupled muscarinic ml receptor and stimulated with carbachol which, unlike antigen, does not induce tyrosine phosphorylation of phospholipase Cfl s. A large difference was observed in the amounts of [3H]inositol phosphates in untreated and herbimycin A-treated cells (>90% decrease) after stimulation with 1 mM carbachol. Values for unstimulated cells were almost identical to those in the Table. When the reduced incor-
8225+90 5547+347 5207_+177 5243-+362
14893+1082 12363+874
hydrolysis. We reemphasize the point that complete accounting of inositol phospholipid and inositol phosphate pools is essential for correct interpretation of all studies with myo-[3H]inositol.
Francesca Santini and Michael A. Beaven
2906+177 2642-+362
Laboratory of Chemical Pharmacology, NHLBI, NIH, Bethesda, MD 20892, USA.
potation of radiolabel i,lto the phospholipid pool was taken into account and values expressed as percent of [3Hlphospholipids in unstimulated cells, the apparent decrease in response was much less (about 50%). Finally, as will be described in detail elsewhere (manuscript in preparation), the residual antigeninduced phospholipid hydrolysis in herbimycin A-treated cells (Table) represented a partially masked G protein-dependent component of
References 1 Keegan, A.D. and Paul, W.E. (1992) Immunol. Today 13, 63-68 2 Beaven, M.A. and Metzger, H. 11993) ImmunoL Today 14, 222-226 3 Uehara, Y., Hori, M., Takeuchi, T. ard Umezawa, H. (1986) MoL Cell. Biol. 6, 2198-2206 4 Lo, T.N., Saul, W. and Beaven, M.A. (1987)]. Biol. Cl:em.262, 4141-4145 5 Jones, S.V.P., Choi, O.H. and Beaven, M.A. (1991) FEBSLetts. 289, 47-50
correctly t°. An analogous methodology was used to predict antigenic sites on proteins j mz. Several statistical methods are Prediction of antigenic sites of available that predict antigenicity proteins is of importance for defin- from the aminoacid sequence based ing antigenic regions of self pro- on the occurrence of a given reins ~ and for the production of aminoacid in an antigenic site 13.t4. synthetic vaccines 2-4. Proteins have These methods were improved by a three-dimensional structure that calculating the accessibility, mobilis determined by their aminoacid ity and hydrophilicity of aminoacid sequence 5. The result of the folding stretches along the polypeptide of the linear polypeptide chain is a chain ~'3. Irrespective of the method minimized energy state for that used for prediction of the antigenic given solute. The protein does so sites, the value is measured by comby compensating charged residues paring the prediction with known with charged or polar solute mol- antigenic sites ts.ts. However, unlike ecules or with other charged side the structure prediction methodolchains of the polypeptide chain. In ogy, no experimental setup is availwater, hydrophobic side chains are able yet that reveals all antigenic buried within the protein to shield sites on a protein. The reliability of them from the solute. Several ele- the prediction depends on the ments of folding exist: ~-helix, 13- knowledge of the antigenic sites of sheet and random coil, the latter the protein and the experimentally has no regular structure and is very obtained antigenic sites that were flexible. Several statistical methods used for evaluating the prediction are now available which can predict methods obtained using Freund's the structure elements of a protein Complete Adjuvant. In a recent from the aminoacid sequencet-L survey in the Netherlands, Predictions are evaluated using the researchers weie asked what adjuthree-dimensional structures as ref- vant they use to evoke an efficient erence. These methods can predict immune response. Approximately up to 56% of the structure elements 75% of the responders used
Freund's Complete Adjuvant. The adjuvant is an oil that is mixed with the antigen and injected into the animal. However, the protein structure in oil is completely different from its structure in a polar solute such as water. Hydrophobic aminoacid side chains will expose their side chain to the oil in a different arrangement to that when the protein is solubilized in water. Thus, the protein will be at least partially denatured. Random coil that does not have a regular structure in water will, in general, not have a regular structure when immersed in oil. It is to be expected that the antibodies obtained using Complete Freund's Adjuvant are directed against structures that are the same in water as in oil, when they are evoked with proteins immersed in oil and screened on proteins solubilized in water. An antibody that is directed towards an a-helix or ~-sheet will not be evoked because, before recognition, the protein structure is destroyed by the oil emulsion. The result will be that those specific B-cells will remain silent. Experimentally determined antigenic sites are thus a restricted set.
A flaw in the detection of antigenic sites
1.m..olo~yTod~y 370
~1. ,4 No. 71993
Why cyclosporin is an effective drug There has been ~, recent spate of articles in Immunology Today concerning the mechanism of action of cyclosporin A (CsA). I am responsible for one and there are two others 1-3. It is riA:y to write about CsA mechanisms because, invariably, before the article appears in print, there are new, surprising findings that require rethinking of any prior hypothesis. All three articles (and others published elsewhere) have focussed on mechanisms by which CsA inhibits the synthesis and secretion of interleukin 2 (IL-2) by T cells. I now sugges'c that the efficacy of CsA as a drug in preventing organ rejection in humans does not rest on that ability. I contend that, in fact, inhibition of IL-2 synthesis can be a deleterious side effect of CsA. It puts the patient in hospital periodically with opportunistic infections. I propose that the key to the success of CsA as a drug in preventing organ rejection, and in the treatment of psoriasis, can be found in ,,,,, recent papers <5 .t_. t,at were
Nonselective actions of herbimycin A We wish to call attention to an unsuspected action of herbimycin A, which is widely used as an inhibitor of tyrosine kinases to define the role of these enzymes in Fc- and T-cell receptor signalling processes 1,-'. Typically, the inhibition of production of inositol phosphates by this reagent is attributed to suppression of tyrosine phosphorylation of phospholipase C vl by a tyrosine kinase. As indicated below, this assumption may not be valid even when herbimycin A does block such phosphorylation. The actions of herbimycin A are apparent only after cells have
added as a note to the proof of my Immunology Today article. They were not available ~lhen the article was submitted. Bc,th papers reported on extracellular, soluble cyclophilins. The Faper by Q. Xu, M.C. Leiva, M.K. Fischkoff et al. s is of particular interest because it reported that 'the extracellular cyclophilin was chemotactic for eosinophils and neutrophils, and that its chemotactic activity was inhibited by CsA. They pointed out that the ubiquitous tissue distribution of cyclophilin (it is present in all cells, even erythrocytes) may reflect the ability of various tissues to respond to injury through the release of cyclophilin resulting in leukocyte infiltration and clearing of the injured cells. Eosinophil infiltration has been associated with both organ graft rejection and psoriasis 3 and CsA is effective in inhibiting the infiltration. Xu et al. suggest that CsA does so by modulating chemotaxis. They propose that this mechanism may be complementary to the other activities of CsA, including inhibition of IL-2 synthesis. I propose that this mechanism, rather than being complementary I • -! to mecnamsms mat involve inhi-
been exposed to it for several hours to allow for complete degradation of the tyrosine kinase 3. Following the protocols of previous wo¢k4, we exposed rat RBL2H3 cells, a tumor analog of rat mucosal mast cells, to 800 aM herbimycin A or vehicle for 14 hours in the presence of growth medium, myo-I3Hlinositol (4 gtCi ml-t) and dinitrophenol-specific lgE, before washing the cells and stimulating them with antigen {20 ng ml-l dinitrophenylated bovine serum albumin) for 15 rain. The Table illustrates a sample experiment in which the incorporation of the inositol into the inositol phospholipid fraction was reduced by >80% in herbimycin-treated ce!ls. The amounts of radiolabeled
Immunology Today
369
bition of IL-2 synthesis, is the prime basis of the success of CsA in prolonging organ graft survival in patients, and in the treatment of psoriasis. It would follow, then, that the search should ,ow be for agents, including CsA derivatives, that strongly inhibit chemotaxis but are less effective in inhibiting iL-2 synthesis by T cells. Bernard F. Erlanger Dept of Microbiology, Columbia University, New York, NY 10032, USA. Re[erences
1 Schreiber, S.L. and Crabtree, G.R. (1992} Immunol. Today 13, 136-142 2 Erlanger, B.F. (1992) Immunol. Today 13, 487-490 3 Wong, R.L., Winslow, C.M. and Cooper, K.D. (1993) Immunol. Today 14, 69-74 4 Sherry, B., Yarlett, N., Strupp, A. and Cerami, A. (1992) Proc. Natl Acad. Sci. USA 89, 3511-3515 5 Xu, Q., LeNa, M.C., Fischkoff, M.K., Handschumacher, R.E. and Lyttle, C.R. (1992)J. BioL Chem. 267, i 1968-i 15~71
inositol in herbimycin A-treated and untreated cells were similar, but the amounts of radiolabeled inositol phospholipids and phosphates were reduced significantly in the treated cells. The distribution of radiolabel among the inositol phospholipid pools (phosphatidylinositol:phosphatidylinositol 4phosphate:phosphatidylinositol 4,5-bisphosphate) in untreated and treated cells, however, was the same namely, 95:2:3 in unstimulated cclis and 90:5:5 in ztimulated cells as determined by thin layer chromatography4. Results such as this indicate that herbimycin A may impede incorporation of inositol into the phospholipid pools. An even more striking result was obtained with RBL-2H3 cells that
vol 14 No. 71993
kaleidoscope letters Treatment
[3H]inositol phosphates
[3H]myoinositol
[3H]inositoi phospholipids
dpm/0.3xl06 cells Untreated
Unstimulated Antigen
450+._27 5994+185
Herbim.-A
Unstimulated Antigen
125_+7 237+-26
had been transfected with the gene for the G protein-coupled muscarinic ml receptor and stimulated with carbachol which, unlike antigen, does not induce tyrosine phosphorylation of phospholipase Cfl s. A large difference was observed in the amounts of [3H]inositol phosphates in untreated and herbimycin A-treated cells (>90% decrease) after stimulation with 1 mM carbachol. Values for unstimulated cells were almost identical to those in the Table. When the reduced incor-
8225+90 5547+347 5207_+177 5243-+362
14893+1082 12363+874
hydrolysis. We reemphasize the point that complete accounting of inositol phospholipid and inositol phosphate pools is essential for correct interpretation of all studies with myo-[3H]inositol.
Francesca Santini and Michael A. Beaven
2906+177 2642-+362
Laboratory of Chemical Pharmacology, NHLBI, NIH, Bethesda, MD 20892, USA.
potation of radiolabel i,lto the phospholipid pool was taken into account and values expressed as percent of [3Hlphospholipids in unstimulated cells, the apparent decrease in response was much less (about 50%). Finally, as will be described in detail elsewhere (manuscript in preparation), the residual antigeninduced phospholipid hydrolysis in herbimycin A-treated cells (Table) represented a partially masked G protein-dependent component of
References 1 Keegan, A.D. and Paul, W.E. (1992) Immunol. Today 13, 63-68 2 Beaven, M.A. and Metzger, H. 11993) ImmunoL Today 14, 222-226 3 Uehara, Y., Hori, M., Takeuchi, T. ard Umezawa, H. (1986) MoL Cell. Biol. 6, 2198-2206 4 Lo, T.N., Saul, W. and Beaven, M.A. (1987)]. Biol. Cl:em.262, 4141-4145 5 Jones, S.V.P., Choi, O.H. and Beaven, M.A. (1991) FEBSLetts. 289, 47-50
correctly t°. An analogous methodology was used to predict antigenic sites on proteins j mz. Several statistical methods are Prediction of antigenic sites of available that predict antigenicity proteins is of importance for defin- from the aminoacid sequence based ing antigenic regions of self pro- on the occurrence of a given reins ~ and for the production of aminoacid in an antigenic site 13.t4. synthetic vaccines 2-4. Proteins have These methods were improved by a three-dimensional structure that calculating the accessibility, mobilis determined by their aminoacid ity and hydrophilicity of aminoacid sequence 5. The result of the folding stretches along the polypeptide of the linear polypeptide chain is a chain ~'3. Irrespective of the method minimized energy state for that used for prediction of the antigenic given solute. The protein does so sites, the value is measured by comby compensating charged residues paring the prediction with known with charged or polar solute mol- antigenic sites ts.ts. However, unlike ecules or with other charged side the structure prediction methodolchains of the polypeptide chain. In ogy, no experimental setup is availwater, hydrophobic side chains are able yet that reveals all antigenic buried within the protein to shield sites on a protein. The reliability of them from the solute. Several ele- the prediction depends on the ments of folding exist: ~-helix, 13- knowledge of the antigenic sites of sheet and random coil, the latter the protein and the experimentally has no regular structure and is very obtained antigenic sites that were flexible. Several statistical methods used for evaluating the prediction are now available which can predict methods obtained using Freund's the structure elements of a protein Complete Adjuvant. In a recent from the aminoacid sequencet-L survey in the Netherlands, Predictions are evaluated using the researchers weie asked what adjuthree-dimensional structures as ref- vant they use to evoke an efficient erence. These methods can predict immune response. Approximately up to 56% of the structure elements 75% of the responders used
Freund's Complete Adjuvant. The adjuvant is an oil that is mixed with the antigen and injected into the animal. However, the protein structure in oil is completely different from its structure in a polar solute such as water. Hydrophobic aminoacid side chains will expose their side chain to the oil in a different arrangement to that when the protein is solubilized in water. Thus, the protein will be at least partially denatured. Random coil that does not have a regular structure in water will, in general, not have a regular structure when immersed in oil. It is to be expected that the antibodies obtained using Complete Freund's Adjuvant are directed against structures that are the same in water as in oil, when they are evoked with proteins immersed in oil and screened on proteins solubilized in water. An antibody that is directed towards an a-helix or ~-sheet will not be evoked because, before recognition, the protein structure is destroyed by the oil emulsion. The result will be that those specific B-cells will remain silent. Experimentally determined antigenic sites are thus a restricted set.
A flaw in the detection of antigenic sites
1.m..olo~yTod~y 370
~1. ,4 No. 71993