Journal of immunological Methods, 148 (1992)255-259
255
© 1992ElsevierSciencePublishersB.V. All rights reserved 0022-1759/92/$05.00
JIM 06294 Commentary
E L I S A kits based on monoclonal antibodies do not measure total IL-I¢I synthesis Charles A. D i n a r e l l o Deportment of Medicine, New England Medical Center and Tufts Unil'ersitySchool of Medicine. Boston. MA 0211i. USA
(Received 15 January 1992) The paper by Herzyk et al. which appears in this issue of the Journal of Immunological Methods is an important and long overdue contribution. The study clearly and convincingly demonstrates that two widely used commercial ELISA kits fail to detect over 90% of the 31 kDa human IL-lfl precursor (pro-IL-lfl) in various cells. This is due to the specificity of the monoclonal antibodies raised to the mature 17 kDa IL-lfl and used in the commercial kits (or in 'home made' ELISAs). The significant amount of pro-IL-l~ which is not measured by the kits casts doubt on the accuracy of total IL-I~ synthesis reported in hundreds of published studies. Investigators evaluating cellular synthesis of IL-1 in the development of new drugs or IL-1 levels in health a~d disease increasingly rely on commercial detection kits. In this commentary, the importance and methods for determining total IL-1B synthesis are reviewed. In view of the preliminary positive results relx,rted on the success of the IL-1 receptor antagonist (IL-lra) in clinical trials, attention should be focased on the amount of IL-1 produced in disease and on the balance of IL-1 versus IL-lra synthesis. Thus, continued use of these ELISA kits which do not detect pro-lL-l~ will confound the interpretation of the IL-1B determinations. Key words: Radioimmunoassay;ELISA; Monoclonalantibody;Interleukin-I/3:lnterleukin-I receptor antagonist
Background The first translation product of IL-1/3 is the 31 kDa precursor (pro-IL-1/3) (Auron et al., 1984) which lacks a clear signal pep'ide; I L - l a lacks a signal peptide as well. Orig~aally described by Gery (Gery et al., 1981), many subsequent studies have reported that a considerable amount of IL-1 remains cell-associated (Auron et al., 1987; Black et al., 1988; Hazuda et al., 1988, 1990; Endres et al., 1989; Lonnemann et al., 1989; Numerof et al., 1990; Rubartelli ct al., 1990; Suttles et al.,
1990a,b). The localization of cell-associated proIL-I~ is almost entirely cytoplasmic, and is not found in the endoplasmic reticulum, Golgi, or plasma membrane fraction (Singer et al., 1988; Beuscher et al., 1990; Rubartelli et al., 1990). Cytosolic IL-1 can be localized to non-clathrin coated vesicles (Rubartelli et al., 1990), microtubules (Baldari and Telford, 1989) or lysosomes (Bakouche et al., 1987). The half-life of cell-associated I L - I , is 15 h whereas that of IL-1//is 2.5 h (Hazuda et al., 1988). Secretion of IL-I[$
Correspondence to: C.A. Dinareno, New England Medical Center, 750 Washington Street, Boston, MA 02111, USA (Tel.: (617)-956-7005;Fax: (617)-956-5292).
Despite the lack of a signal peptide, IL-1// does get out of the cell. The amount of IL-I
256 which is "secreted" depends upon the cell type and the conditions of stimulation. For example, the human peripheral blood monocyte appears to be highly efficient in secreting IL-lfl compared to endothelial cells, smooth muscle cells and fibroblasts. Between 35 and 70% of IL-1/3 is secreted from human peripheral blood mononuclear cells (PBMC) when stimulated with low concentrations of LPS whereas under the same condition, nearly all I L - l a remains cell-associated (Endres et al., 1989; Lonnemann et al., 1989). Using heat-killed Staphylococcus epidermidis as a stimulus, almost all IL-lfl is secreted from PBMC. In contrast to stimulation with bacteria or LPS, when PBMC are stimulated with I L - l a or IL-2, nearly all IL-lfi remains cell-associated (Dinarello et al., 1987; Numerof et al., 1988, 1990). It is still unclear how IL-1 is transported from the cytosol to the extracellular compartment, but recent evidence suggests that a multiple drug resistance glycoprotein may be involved in this event (Young and Krasney, 1991). Secretion and processing to the mature peptide appear to be linked events, although some studies demonstrate that pro-IL-lfl can be secreted intact (Auron et al., 1987) and then later cleaved by serine proteases present in inflamed tissue.
Cleavage of IL-I~
Mature IL-lfl has an N terminus at the alanine position 117 (Van Damme et al., 1985) but other naturally occurring N termini have been reported (Knudsen et al., 1986; Mizutani et al., 1991). A 22 kDa intermediate peptide is found in the supernatants of monocytes (Auron et al., 1987; Beuscher et al., 1990), suggesting that the pro-ILl/3 can be secreted prior to generation of the mature peptide. Cell injury certainly contributes to the release of pro-IL-1/3. Elastase, plasmin, cathepsin G, collagenase, and other serine proteases, as well as surface enkephalinase, have been implicated in the cleavage of pro-IL-lfl into its 17.5 kDa mature carboxyl fragment (reviewed in Dinarello, 1991). A monocyte-specific protease has been described that specifically cleaves IL-lfl at the alanine position (Black et al., 1988; Kostura et al., 1989). This
protease is not found in fibroblasts but rather in monocytes and monocyte cell lines. Recent studies have identified and purified the IL-1 cleavage protease which has resulted in the cloning of a cDNA (Cerretti et al., 1991). When transfected into cells, the eDNA codes for an IL-1 cleavage enzyme (convertase) which cuts pro-IL-lfl at the alanine position (Cerretti et al., 1991). A dermal mast cell kinase also accomplishes the cleavage (Mizutani et al., 1991). Blockade of specific proIL-1 processing enzymes has been proposed as a strategy for preventing the effects of IL-1 in discase. The appearance of mature and smaller molecular weight IL-1 peptides can be prevented by inhibitors of serine proteases. These smaller molecular weight peptides which are detected by bioassay following gel filtration (Cannon and Dinarello, 1985) are routinely found in human plasma, urine, and peritoneal, pleural, and joint fluids. The fragments are likely cleaved via trypsin sensitive sites and human IL-lfl contains several cleavage sites for serine proteases (Auron et al., 1987; Dinarello, 1988). The biologic authenticity of these fragments of mature IL-lfl has been demonstrated using neutralizing polyclonal antibodies raised against the recombinant human IL1/3. In preparations of recombinant IL-1/3, we have identified a 5488 Da C terminal peptide (Dinarello, 1988), generated at the lysine-lysineiysine site of human IL-1/3, which could represent an active C terminal fragment. Other studies have shown that a synthetic peptide in this region (208-240) is biologically active (Obal et ai., 1990) and blocked by the IL-lra (Opp et al., 1992). Other synthetic peptides of human IL-lfl possess immunostimulatory but not inflammatory properties (Boraschi et al., 1990).
How to measure pro-IL.11~?
The studies by Herzyk et al. clearly demonstrate that epitopes of native pro-IL-lfl are not recognized by the monoclonal antibodies raised to the mature form whereas denatured pro-IL-1/3 undergoes conformational changes which expose these epitopes, in contrast, polyclonal antisera raised to the mature form of IL-lfl contain anti-
257 bodies which detect the native pro-IL-1/3. Investigators using radioimmunoassays based on polyclonal antibodies to mature IL-lfl have demonstrated that both mature as well as native pro-ILlfl are quantitatively measured (Numerof et al., 1988, 1990; Schindler et al., 1990a). Specifically denaturing the samples intended for IL-lfl ELISA determinations has not been studied.
Why measure total 11.-113 synthesis? Why is measurement of the synthesis of the primary translational product as well as any processed product meaningful? Some will argue that it is only the mature, biologically active IL-lfl which is relevant to measure, not the precursor. This author disagrees for several reasons. Total IL-1/3 protein synthesis reflects transcriptional and translational control of the IL-1/3 gene under the pathologic conditions being studied. In contrast, post-translational processing of in vitro production of IL-I is vulnerable to cell culture artifacts and is quite removed from clinical situations. For example, in sites of infection or inflammation, cell death and the release of neutrophil elastase is a fundamental event but most laboratory culture conditions avoid cell injury and do not contain activated neutrophils. The cells are cultured in small amounts of serum which is usually heated and often of fetal origin. Serum itself is a laboratory artifact devoid of several substances which enhance processing. Isolation (Ficoll-Hypaque) techniques, crowding of large numbers of cells, adherence to glass or polystyrene plastic surfaces are not encountered in vivo. One condition for cytokine production which approaches the natural state of circulating blood cells is rotating or pumping whole, heparinized blood (Bingel et al., 1986; Schindler et al., 1990b; Poutsiaka et al., 1991). For example, the amount of IL-1/3 synthesized in rotating cultures is 50% greater than synthesis in stationary cultures (Poutsiaka et al., 1991) The vast majority of studies on human cytokine production employ the in vitro culture of PBMCs or monocytes. Many investigators stimulate these cells with LPS concentrations as high as 10 # g / m l whereas 10-100 pg/ml is closer to
the clinical situation in Gram-negative sepsis. Using these high concentrations of stimulants, more IL-I/3 is secreted and processed to the mature form. This manipulation of cell culture conditions to yield a greater proportion of mature 11--1/3 runs the risk of being further removed from the clinical situation. If one wants to know how much IL-I/3 is being synthesized in a disease, or in response to a drug, one would have to know the amount of the primary translational product as well as the amount of mature peptide, that is, both need to be measured. Since the commercial kits only measure the mature peptide, a considerable amount of IL-I~ synthesis has gone undetected.
Responsibility of kit manufacturers In general, the kit makers produce highly sensitive and rapid assays. However, their sensitivity claims are based on recombinant cytokine standards, rarely on natural forms of the cytokines. In the case of IL-1/L a standard using the natural pro-lL-1/3 is a case in point as demonstrated by the Herzyk paper. Investigators should be critical of all assay kit and carry out their own controls. A new problem is brewing as kit makers for TNF and IL-6 have not evaluated the effect of these cytokine determinations in the presence of soluble receptors or binding proteins which are clearly present in cell cultures and body fluids (Engelmann et al., 1989; Novick et al., 1989) and may confound the assays. Furthermore, investigators should question the accuracy of 1I.,-1/3 kits which employ monoclonal antibodies to the mature peptide. It is possible that a similar situation may also exist for human IL-la. The consequences for using less than accurate kits are far reaching and require that data must be re-interpreted, concepts re-evaluted and possibly some experiments repeated.
Acknowledgements I thank Drs. B.D. Clark, J.G. Cannon, E.V. Granowitz, L.C. Miller, D.D. Poutsiaka, R. Roubenoff, and L. Shapiro for helpful sugges-
258 tions. Special thanks to Dr. Sheldon M. Wolff for his c r i t i c a l r e v i e w o f t h e m a n u s c r i p t .
References Auron. P.E., Webb, A.C., Rosenwasser, L.J., Mucci, S.F., Rich, A., Wolff. S.M. and Dinarello, C.A. (1984) Nueleotide sequence of human monocyte interleukin 1 precursor eDNA. Proc. Natl. Acad. Sci. USA 81, 7907-7911. Auron, P.E.. Warner, S.J., Webb, A.C., Cannon, J.G., Bernheim, H.A., McAdam, K.J., Rosenwasser, L.J., LoPreste, G., Mucci. S.F. and Dinarello, C.A. (1987) Studies on the molecular nature of human interleukin 1. J. lmmunol. 138, 1447-1456. Bakouche, O., Brown, D.C. and Lachman, LB. (1987) Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release. J. immunol. 138, 4249-4255. Baldari, C.T. and Telford, J.L. (1989) The intracellular precursor of IL-I/3 is associated with microtubules in activated U937 cells. J. Immunol. 142, 785-791. Beuscher, H.U., Guenther, C. and Roellinghoff, M. (1990) 1L-I beta is secreted by activated murine macrophagcs as biologically inactive precursor. J. lmmunol. 144, 21792183. Bingel, M., Lonnemann, G., Shaldon, S., Koch, K.M. and Dinarello, C.A. (1986) Human interlcukin-1 production during hemodialysis. Nephron 43, 161-163. Black, R.A., Kronheim, S.R., Cantrell, M., Deeley, M.C.. March, C.J.. Prickett, K.S.. Wignall, J., Conlon, P.J., Cosman, D. and Hopp, T.P. (1988) Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor. J. Biol. Chem. 263, 9437-9442. Boraschi, D., Antoni, G., Perni, F., Villa, L., Nencioni, L., Ghiara, P., Presentini, R. and Tagliabue, A. (1990) Defining the structural requirements of a biologially active domain of human IL-1/L 1, 21-26. Cannon, J.G. and Dinarello, C.A. (1985) Increased plasma interleukin-I activity in women after ovulation. Science 227, 1247-1249. Cerretti, D.P., Mosley, B., Kozlosky, C., Nelson, N., Van Ness, K., Greenstreet, T., Sleath, P.R., March, C.J., Kronheim, S.R. and Black, R.A. (1991) Molecular cloning of the IL-1/3 processing enzyme, abstract Cytokine 3, 472. Dinarello, C.A. (1988) Biology of interleukin 1. FASEB J. 2, 108-115. Dinarello, C.A. (1991) lnterleukin-I and interleukin-I antagonism. Blood 77, 1627-1652. Dinarello. C.A., Ikejima. T., Warner, S.J., Orencole, S.F., Lonnemann, G., Cannon, J.G. and Libby, P. (1987) Interleukin 1 induces interleukin 1. I. Induction of circulating interleukin 1 in rabbits in vivo and in human mononuclear cells in vitro. J Immunol. 139, 1902-1910. Endres, S., Cannon, J.G., Ghorbani, R., Dempsey, R.A., Sisson. S.D., Lonnemann, G., Van der Meet, J.W.M., Wolff. S.M. and Dinarello, C.A. (1989) In vitro production of IL-lbeta, IL-lalpha, TNF, and IL-2 in healthy subjects:
distribution, effect of cyclooxygenase inhibition and evidence of independent gene regulation. Eur J Immunol. 19, 2327-2333. Engelmann. H., Aderka, D., Rubinstein, M., Rotman, D. and Wallach, D. (1989) A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity. J. Biol. Chem. 264, 11974-11980. Gery, 1., Davies, P., Derr, J., Krett, N. and Barranger, J.A. (1981) Relationship between production and release of lymphocyte ac:i'~ating factor (interleukin-l) by murine macrophages: effects of various agents. Cell. Immunol. 64, 293-303. Hazuda, D.J., Lee, J.C. and Young, P.R. (1988) The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta. J. Biol. Chem. 263, 8473-8479. Hazuda, D.J., Strickler, J., Kueppers, F., Simon, P.L. and Young, P.R. (1990) Processing of precursor interleukin-I beta and inflammatory disease. J. Biol. Chem. 265, 63186322. Knudsen, P.J., Dinarello, C.A. and Strom, T.B. (1986) Purification and characterization of a unique human interleukin I from the tumor cell line U937. J. Immunol. 136, 33113316. Kostura, M.J., Tocci, MJ., Limjuco, G., Chin, J., Cameron, P., Hillman, A.G., Chartrain, N.A. and Schmidt, J.A. (1989) Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proc. Natl. Acad. Sci. USA 86, 52275231. Lonnemann, G., Endres, S., van der Meer, J.W., Cannon, J.G., Koch, K.M. and Dinarello, C.A. (1989) Differences in the synthesis and kinetics of release of interleukin I alpha, interleukin 1 beta and tumor necrosis factor from human mononuclear cells. Eur. J. lmmunol. 19,1531-1536. Mizutani, H., Schecter, N., Zazarus, G., Black, R.A. and Kupper, T.S. (1991) Rapid and specific conversion of precursor interleukin-lb to an active IL-1 species by human mast cell chymase. J, Exp. Med. 174, 821-825. Novick, D., Engelmann, H., Wallach, D. and Rubinstein, M. (1989) Soluble cytokine receptors are present in normal human urine. J. Exp. Med. 170, 1409-1414. Numerof, R.P., Aronson, F.R. and Mier, J.W. (1988) IL-2 stimulates the production of IL-la and IL-I/3 by human peripheral blood mononuclear cells. J. lmmunol. 141, 4250-4257. Numerof, R.P., Kotik, A.N., Dinarello, C.A. and Mier, J.W. (1990) Pro-interleukin-I beta production by a subpopulation of human T cells, but not NK cells, in response to interleukin-2. Cell Immunol. 130, 118-128. Obal, F., Opp, M., Cady, A.B., Johannsen, L., Postlethwaite, A.E., Poppleton, H.M., Seyer, J.M. and Krueger, J.M. (1990) Interleukin-la and an 1L-I/3 fragment are somnogenie. Am. J. Physiol. 259, R ~.39-R446. Opp, M.R., Postlethwaite, A.E., Seyer, J.M. and Krueger, J.M. (1992) Interleukin-I receptor antagonist blocks somnogenie and pyrogenic responses to an IL-I fragment, Proc. Natl. Acad. Sci. USA, in press. Poutsiaka, D.D., Clark, B.D., Vannier, E. and Dinarello, C.A,
259 (1991) Production of interleukin-I receptor antagonist and interleukin-I/3 by peripheral blood mononuclear cells is differentially regulated. Blood 78. 1275- t 281. Ruhartelli, A., Cozzolino, F.. Talio, M. and Sitia, K. (1990) A novel secretory pathway for interleukin-t ~he'.a, a protein lacking a signal sequence. Emho J. 9, 1503-1510. Schindler, R., Clark, B.D. and Dinarello, C.A. (1990a) Dissociation between interleukin-I/3 mRNA and protein synthesis in human peripheral blood mononuclear cells. J. Biol. Chem. 265, 10232-10237. Schindler, R., Lonnemann, G., Shaldon, S., Koch, K.M. and Dinarello, C.A. (1990b) Transcription, not synthesis, of interleukin-I and tumor necrosis factor by complement. Kidney Int. 37, 85-93. Singer, I.I., Scott, S., Hall, G.L., Limjuco, G., Chin, J. and Schmidt, J.A. (1988) lnterleukin l beta is localized in the cytoplasmic ground substance but is largely absent from
the Golgi apparatus and plasma membranes of stimulated human monocytes. J. Exp. Med. 167, 389-4[}7. Suttles, J., Carruth, L.M. and Mizel, S.B. (1990a) Detection of IL-Ic~ and IL-I/3 in the supernatants of paraformaldehyde-treated human mon(~cy~.es.Evidence against a membrane form of IL-I. J. Immunol. 144, 170-174. Suttlcs, J., Girl J.G. and Mizel, S.B. (l{Y'~'}b)IL-I secretion by macrophages. Enhancement of IL-I secretion and processing by calcium ionophores. ,1. Immunol. 144, 175-182. Van Damme, J., De Ley, M., Opdenakker, G., Billiau, A. and De Somer, P. (1985) Homogeneous interferon-inducing 22K h c t o r is related to endogenous pyrogen and interlcukin-I. Nature 314, 266-268. Young, P.R. and Krasney, P.A. (1991) interleukin-l/3 can be secreted from mammalian cells by the human MDRI P-glycoprotein, (abstract) Cytokine 3, 472.
255
© 1992ElsevierSciencePublishersB.V. All rights reserved 0022-1759/92/$05.00
JIM 06294 Commentary
E L I S A kits based on monoclonal antibodies do not measure total IL-I¢I synthesis Charles A. D i n a r e l l o Deportment of Medicine, New England Medical Center and Tufts Unil'ersitySchool of Medicine. Boston. MA 0211i. USA
(Received 15 January 1992) The paper by Herzyk et al. which appears in this issue of the Journal of Immunological Methods is an important and long overdue contribution. The study clearly and convincingly demonstrates that two widely used commercial ELISA kits fail to detect over 90% of the 31 kDa human IL-lfl precursor (pro-IL-lfl) in various cells. This is due to the specificity of the monoclonal antibodies raised to the mature 17 kDa IL-lfl and used in the commercial kits (or in 'home made' ELISAs). The significant amount of pro-IL-l~ which is not measured by the kits casts doubt on the accuracy of total IL-I~ synthesis reported in hundreds of published studies. Investigators evaluating cellular synthesis of IL-1 in the development of new drugs or IL-1 levels in health a~d disease increasingly rely on commercial detection kits. In this commentary, the importance and methods for determining total IL-1B synthesis are reviewed. In view of the preliminary positive results relx,rted on the success of the IL-1 receptor antagonist (IL-lra) in clinical trials, attention should be focased on the amount of IL-1 produced in disease and on the balance of IL-1 versus IL-lra synthesis. Thus, continued use of these ELISA kits which do not detect pro-lL-l~ will confound the interpretation of the IL-1B determinations. Key words: Radioimmunoassay;ELISA; Monoclonalantibody;Interleukin-I/3:lnterleukin-I receptor antagonist
Background The first translation product of IL-1/3 is the 31 kDa precursor (pro-IL-1/3) (Auron et al., 1984) which lacks a clear signal pep'ide; I L - l a lacks a signal peptide as well. Orig~aally described by Gery (Gery et al., 1981), many subsequent studies have reported that a considerable amount of IL-1 remains cell-associated (Auron et al., 1987; Black et al., 1988; Hazuda et al., 1988, 1990; Endres et al., 1989; Lonnemann et al., 1989; Numerof et al., 1990; Rubartelli ct al., 1990; Suttles et al.,
1990a,b). The localization of cell-associated proIL-I~ is almost entirely cytoplasmic, and is not found in the endoplasmic reticulum, Golgi, or plasma membrane fraction (Singer et al., 1988; Beuscher et al., 1990; Rubartelli et al., 1990). Cytosolic IL-1 can be localized to non-clathrin coated vesicles (Rubartelli et al., 1990), microtubules (Baldari and Telford, 1989) or lysosomes (Bakouche et al., 1987). The half-life of cell-associated I L - I , is 15 h whereas that of IL-1//is 2.5 h (Hazuda et al., 1988). Secretion of IL-I[$
Correspondence to: C.A. Dinareno, New England Medical Center, 750 Washington Street, Boston, MA 02111, USA (Tel.: (617)-956-7005;Fax: (617)-956-5292).
Despite the lack of a signal peptide, IL-1// does get out of the cell. The amount of IL-I
256 which is "secreted" depends upon the cell type and the conditions of stimulation. For example, the human peripheral blood monocyte appears to be highly efficient in secreting IL-lfl compared to endothelial cells, smooth muscle cells and fibroblasts. Between 35 and 70% of IL-1/3 is secreted from human peripheral blood mononuclear cells (PBMC) when stimulated with low concentrations of LPS whereas under the same condition, nearly all I L - l a remains cell-associated (Endres et al., 1989; Lonnemann et al., 1989). Using heat-killed Staphylococcus epidermidis as a stimulus, almost all IL-lfl is secreted from PBMC. In contrast to stimulation with bacteria or LPS, when PBMC are stimulated with I L - l a or IL-2, nearly all IL-lfi remains cell-associated (Dinarello et al., 1987; Numerof et al., 1988, 1990). It is still unclear how IL-1 is transported from the cytosol to the extracellular compartment, but recent evidence suggests that a multiple drug resistance glycoprotein may be involved in this event (Young and Krasney, 1991). Secretion and processing to the mature peptide appear to be linked events, although some studies demonstrate that pro-IL-lfl can be secreted intact (Auron et al., 1987) and then later cleaved by serine proteases present in inflamed tissue.
Cleavage of IL-I~
Mature IL-lfl has an N terminus at the alanine position 117 (Van Damme et al., 1985) but other naturally occurring N termini have been reported (Knudsen et al., 1986; Mizutani et al., 1991). A 22 kDa intermediate peptide is found in the supernatants of monocytes (Auron et al., 1987; Beuscher et al., 1990), suggesting that the pro-ILl/3 can be secreted prior to generation of the mature peptide. Cell injury certainly contributes to the release of pro-IL-1/3. Elastase, plasmin, cathepsin G, collagenase, and other serine proteases, as well as surface enkephalinase, have been implicated in the cleavage of pro-IL-lfl into its 17.5 kDa mature carboxyl fragment (reviewed in Dinarello, 1991). A monocyte-specific protease has been described that specifically cleaves IL-lfl at the alanine position (Black et al., 1988; Kostura et al., 1989). This
protease is not found in fibroblasts but rather in monocytes and monocyte cell lines. Recent studies have identified and purified the IL-1 cleavage protease which has resulted in the cloning of a cDNA (Cerretti et al., 1991). When transfected into cells, the eDNA codes for an IL-1 cleavage enzyme (convertase) which cuts pro-IL-lfl at the alanine position (Cerretti et al., 1991). A dermal mast cell kinase also accomplishes the cleavage (Mizutani et al., 1991). Blockade of specific proIL-1 processing enzymes has been proposed as a strategy for preventing the effects of IL-1 in discase. The appearance of mature and smaller molecular weight IL-1 peptides can be prevented by inhibitors of serine proteases. These smaller molecular weight peptides which are detected by bioassay following gel filtration (Cannon and Dinarello, 1985) are routinely found in human plasma, urine, and peritoneal, pleural, and joint fluids. The fragments are likely cleaved via trypsin sensitive sites and human IL-lfl contains several cleavage sites for serine proteases (Auron et al., 1987; Dinarello, 1988). The biologic authenticity of these fragments of mature IL-lfl has been demonstrated using neutralizing polyclonal antibodies raised against the recombinant human IL1/3. In preparations of recombinant IL-1/3, we have identified a 5488 Da C terminal peptide (Dinarello, 1988), generated at the lysine-lysineiysine site of human IL-1/3, which could represent an active C terminal fragment. Other studies have shown that a synthetic peptide in this region (208-240) is biologically active (Obal et ai., 1990) and blocked by the IL-lra (Opp et al., 1992). Other synthetic peptides of human IL-lfl possess immunostimulatory but not inflammatory properties (Boraschi et al., 1990).
How to measure pro-IL.11~?
The studies by Herzyk et al. clearly demonstrate that epitopes of native pro-IL-lfl are not recognized by the monoclonal antibodies raised to the mature form whereas denatured pro-IL-1/3 undergoes conformational changes which expose these epitopes, in contrast, polyclonal antisera raised to the mature form of IL-lfl contain anti-
257 bodies which detect the native pro-IL-1/3. Investigators using radioimmunoassays based on polyclonal antibodies to mature IL-lfl have demonstrated that both mature as well as native pro-ILlfl are quantitatively measured (Numerof et al., 1988, 1990; Schindler et al., 1990a). Specifically denaturing the samples intended for IL-lfl ELISA determinations has not been studied.
Why measure total 11.-113 synthesis? Why is measurement of the synthesis of the primary translational product as well as any processed product meaningful? Some will argue that it is only the mature, biologically active IL-lfl which is relevant to measure, not the precursor. This author disagrees for several reasons. Total IL-1/3 protein synthesis reflects transcriptional and translational control of the IL-1/3 gene under the pathologic conditions being studied. In contrast, post-translational processing of in vitro production of IL-I is vulnerable to cell culture artifacts and is quite removed from clinical situations. For example, in sites of infection or inflammation, cell death and the release of neutrophil elastase is a fundamental event but most laboratory culture conditions avoid cell injury and do not contain activated neutrophils. The cells are cultured in small amounts of serum which is usually heated and often of fetal origin. Serum itself is a laboratory artifact devoid of several substances which enhance processing. Isolation (Ficoll-Hypaque) techniques, crowding of large numbers of cells, adherence to glass or polystyrene plastic surfaces are not encountered in vivo. One condition for cytokine production which approaches the natural state of circulating blood cells is rotating or pumping whole, heparinized blood (Bingel et al., 1986; Schindler et al., 1990b; Poutsiaka et al., 1991). For example, the amount of IL-1/3 synthesized in rotating cultures is 50% greater than synthesis in stationary cultures (Poutsiaka et al., 1991) The vast majority of studies on human cytokine production employ the in vitro culture of PBMCs or monocytes. Many investigators stimulate these cells with LPS concentrations as high as 10 # g / m l whereas 10-100 pg/ml is closer to
the clinical situation in Gram-negative sepsis. Using these high concentrations of stimulants, more IL-I/3 is secreted and processed to the mature form. This manipulation of cell culture conditions to yield a greater proportion of mature 11--1/3 runs the risk of being further removed from the clinical situation. If one wants to know how much IL-I/3 is being synthesized in a disease, or in response to a drug, one would have to know the amount of the primary translational product as well as the amount of mature peptide, that is, both need to be measured. Since the commercial kits only measure the mature peptide, a considerable amount of IL-I~ synthesis has gone undetected.
Responsibility of kit manufacturers In general, the kit makers produce highly sensitive and rapid assays. However, their sensitivity claims are based on recombinant cytokine standards, rarely on natural forms of the cytokines. In the case of IL-1/L a standard using the natural pro-lL-1/3 is a case in point as demonstrated by the Herzyk paper. Investigators should be critical of all assay kit and carry out their own controls. A new problem is brewing as kit makers for TNF and IL-6 have not evaluated the effect of these cytokine determinations in the presence of soluble receptors or binding proteins which are clearly present in cell cultures and body fluids (Engelmann et al., 1989; Novick et al., 1989) and may confound the assays. Furthermore, investigators should question the accuracy of 1I.,-1/3 kits which employ monoclonal antibodies to the mature peptide. It is possible that a similar situation may also exist for human IL-la. The consequences for using less than accurate kits are far reaching and require that data must be re-interpreted, concepts re-evaluted and possibly some experiments repeated.
Acknowledgements I thank Drs. B.D. Clark, J.G. Cannon, E.V. Granowitz, L.C. Miller, D.D. Poutsiaka, R. Roubenoff, and L. Shapiro for helpful sugges-
258 tions. Special thanks to Dr. Sheldon M. Wolff for his c r i t i c a l r e v i e w o f t h e m a n u s c r i p t .
References Auron. P.E., Webb, A.C., Rosenwasser, L.J., Mucci, S.F., Rich, A., Wolff. S.M. and Dinarello, C.A. (1984) Nueleotide sequence of human monocyte interleukin 1 precursor eDNA. Proc. Natl. Acad. Sci. USA 81, 7907-7911. Auron, P.E.. Warner, S.J., Webb, A.C., Cannon, J.G., Bernheim, H.A., McAdam, K.J., Rosenwasser, L.J., LoPreste, G., Mucci. S.F. and Dinarello, C.A. (1987) Studies on the molecular nature of human interleukin 1. J. lmmunol. 138, 1447-1456. Bakouche, O., Brown, D.C. and Lachman, LB. (1987) Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release. J. immunol. 138, 4249-4255. Baldari, C.T. and Telford, J.L. (1989) The intracellular precursor of IL-I/3 is associated with microtubules in activated U937 cells. J. Immunol. 142, 785-791. Beuscher, H.U., Guenther, C. and Roellinghoff, M. (1990) 1L-I beta is secreted by activated murine macrophagcs as biologically inactive precursor. J. lmmunol. 144, 21792183. Bingel, M., Lonnemann, G., Shaldon, S., Koch, K.M. and Dinarello, C.A. (1986) Human interlcukin-1 production during hemodialysis. Nephron 43, 161-163. Black, R.A., Kronheim, S.R., Cantrell, M., Deeley, M.C.. March, C.J.. Prickett, K.S.. Wignall, J., Conlon, P.J., Cosman, D. and Hopp, T.P. (1988) Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor. J. Biol. Chem. 263, 9437-9442. Boraschi, D., Antoni, G., Perni, F., Villa, L., Nencioni, L., Ghiara, P., Presentini, R. and Tagliabue, A. (1990) Defining the structural requirements of a biologially active domain of human IL-1/L 1, 21-26. Cannon, J.G. and Dinarello, C.A. (1985) Increased plasma interleukin-I activity in women after ovulation. Science 227, 1247-1249. Cerretti, D.P., Mosley, B., Kozlosky, C., Nelson, N., Van Ness, K., Greenstreet, T., Sleath, P.R., March, C.J., Kronheim, S.R. and Black, R.A. (1991) Molecular cloning of the IL-1/3 processing enzyme, abstract Cytokine 3, 472. Dinarello, C.A. (1988) Biology of interleukin 1. FASEB J. 2, 108-115. Dinarello, C.A. (1991) lnterleukin-I and interleukin-I antagonism. Blood 77, 1627-1652. Dinarello. C.A., Ikejima. T., Warner, S.J., Orencole, S.F., Lonnemann, G., Cannon, J.G. and Libby, P. (1987) Interleukin 1 induces interleukin 1. I. Induction of circulating interleukin 1 in rabbits in vivo and in human mononuclear cells in vitro. J Immunol. 139, 1902-1910. Endres, S., Cannon, J.G., Ghorbani, R., Dempsey, R.A., Sisson. S.D., Lonnemann, G., Van der Meet, J.W.M., Wolff. S.M. and Dinarello, C.A. (1989) In vitro production of IL-lbeta, IL-lalpha, TNF, and IL-2 in healthy subjects:
distribution, effect of cyclooxygenase inhibition and evidence of independent gene regulation. Eur J Immunol. 19, 2327-2333. Engelmann. H., Aderka, D., Rubinstein, M., Rotman, D. and Wallach, D. (1989) A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity. J. Biol. Chem. 264, 11974-11980. Gery, 1., Davies, P., Derr, J., Krett, N. and Barranger, J.A. (1981) Relationship between production and release of lymphocyte ac:i'~ating factor (interleukin-l) by murine macrophages: effects of various agents. Cell. Immunol. 64, 293-303. Hazuda, D.J., Lee, J.C. and Young, P.R. (1988) The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta. J. Biol. Chem. 263, 8473-8479. Hazuda, D.J., Strickler, J., Kueppers, F., Simon, P.L. and Young, P.R. (1990) Processing of precursor interleukin-I beta and inflammatory disease. J. Biol. Chem. 265, 63186322. Knudsen, P.J., Dinarello, C.A. and Strom, T.B. (1986) Purification and characterization of a unique human interleukin I from the tumor cell line U937. J. Immunol. 136, 33113316. Kostura, M.J., Tocci, MJ., Limjuco, G., Chin, J., Cameron, P., Hillman, A.G., Chartrain, N.A. and Schmidt, J.A. (1989) Identification of a monocyte specific pre-interleukin 1 beta convertase activity. Proc. Natl. Acad. Sci. USA 86, 52275231. Lonnemann, G., Endres, S., van der Meer, J.W., Cannon, J.G., Koch, K.M. and Dinarello, C.A. (1989) Differences in the synthesis and kinetics of release of interleukin I alpha, interleukin 1 beta and tumor necrosis factor from human mononuclear cells. Eur. J. lmmunol. 19,1531-1536. Mizutani, H., Schecter, N., Zazarus, G., Black, R.A. and Kupper, T.S. (1991) Rapid and specific conversion of precursor interleukin-lb to an active IL-1 species by human mast cell chymase. J, Exp. Med. 174, 821-825. Novick, D., Engelmann, H., Wallach, D. and Rubinstein, M. (1989) Soluble cytokine receptors are present in normal human urine. J. Exp. Med. 170, 1409-1414. Numerof, R.P., Aronson, F.R. and Mier, J.W. (1988) IL-2 stimulates the production of IL-la and IL-I/3 by human peripheral blood mononuclear cells. J. lmmunol. 141, 4250-4257. Numerof, R.P., Kotik, A.N., Dinarello, C.A. and Mier, J.W. (1990) Pro-interleukin-I beta production by a subpopulation of human T cells, but not NK cells, in response to interleukin-2. Cell Immunol. 130, 118-128. Obal, F., Opp, M., Cady, A.B., Johannsen, L., Postlethwaite, A.E., Poppleton, H.M., Seyer, J.M. and Krueger, J.M. (1990) Interleukin-la and an 1L-I/3 fragment are somnogenie. Am. J. Physiol. 259, R ~.39-R446. Opp, M.R., Postlethwaite, A.E., Seyer, J.M. and Krueger, J.M. (1992) Interleukin-I receptor antagonist blocks somnogenie and pyrogenic responses to an IL-I fragment, Proc. Natl. Acad. Sci. USA, in press. Poutsiaka, D.D., Clark, B.D., Vannier, E. and Dinarello, C.A,
259 (1991) Production of interleukin-I receptor antagonist and interleukin-I/3 by peripheral blood mononuclear cells is differentially regulated. Blood 78. 1275- t 281. Ruhartelli, A., Cozzolino, F.. Talio, M. and Sitia, K. (1990) A novel secretory pathway for interleukin-t ~he'.a, a protein lacking a signal sequence. Emho J. 9, 1503-1510. Schindler, R., Clark, B.D. and Dinarello, C.A. (1990a) Dissociation between interleukin-I/3 mRNA and protein synthesis in human peripheral blood mononuclear cells. J. Biol. Chem. 265, 10232-10237. Schindler, R., Lonnemann, G., Shaldon, S., Koch, K.M. and Dinarello, C.A. (1990b) Transcription, not synthesis, of interleukin-I and tumor necrosis factor by complement. Kidney Int. 37, 85-93. Singer, I.I., Scott, S., Hall, G.L., Limjuco, G., Chin, J. and Schmidt, J.A. (1988) lnterleukin l beta is localized in the cytoplasmic ground substance but is largely absent from
the Golgi apparatus and plasma membranes of stimulated human monocytes. J. Exp. Med. 167, 389-4[}7. Suttles, J., Carruth, L.M. and Mizel, S.B. (1990a) Detection of IL-Ic~ and IL-I/3 in the supernatants of paraformaldehyde-treated human mon(~cy~.es.Evidence against a membrane form of IL-I. J. Immunol. 144, 170-174. Suttlcs, J., Girl J.G. and Mizel, S.B. (l{Y'~'}b)IL-I secretion by macrophages. Enhancement of IL-I secretion and processing by calcium ionophores. ,1. Immunol. 144, 175-182. Van Damme, J., De Ley, M., Opdenakker, G., Billiau, A. and De Somer, P. (1985) Homogeneous interferon-inducing 22K h c t o r is related to endogenous pyrogen and interlcukin-I. Nature 314, 266-268. Young, P.R. and Krasney, P.A. (1991) interleukin-l/3 can be secreted from mammalian cells by the human MDRI P-glycoprotein, (abstract) Cytokine 3, 472.
