ACTTVATTON OF TRANSCRIPTION FACTOR COMPLEX BY /?(l-3) GLUCAN
BACKGROUND OF THE INVENTION
Underivatized, aqueous soluble β (1-3 ) -glucan (also known as PGG-Glucan or BETAFECTIN®) is a novel and unique soluble β-glucan manufactured through a proprietary process. The biological activity of this molecule is clearly distinguishable from particulate or other soluble β-glucans. Numerous laboratories have reported direct induction of arachidonic acid metabolites (Czop et al . , J. Immunol . 141 (9) : 3170-3176 (1988)), cytokines (Abel and Czop, Intl . J. Immunopharmacol . , 14 (8) : 1363-1373 (1992); Doita et al . , J. Leuk . Biol . 14(2) .173-183 (1991)) and oxidative burst (Cain et al . , Complement 4:75-86 (1987); Gallin et al . , Int . J. Immunopharmacol . 14 (2) : 173 -183
(1992)) by both particulate and soluble forms of β-glucans. In contrast, underivatized, aqueous soluble β (1-3) -glucan does not directly activate leukocyte functions such as oxidative burst activity (Mackin et al . , FASEB J. 8:A216 (1994)), cytokine secretion (Putsiaka et al . , Blood
82:3695-3700 (1993)) or proliferation ( akshull et al . , J. Cell . Biochem . suppl . 18A -. 22 (1994)) . Instead, underivatized, aqueous soluble β (1-3 ) -glucan primes cells for activation by secondary stimuli (Mackin et al . (1994); Brunke-Reese and Mackin, FASEB J. 8:A488 (1994) ; and Wakshull, E. et al . , (1994)) . The mechanism by which aqueous soluble β (1-3 ) -glucan activates or primes these cellular functions is currently unknown.
SUMMARY OF THE INVENTION This invention pertains to the discovery that underivatized, aqueous soluble β (1-3 ) -glucan specifically increases activation of a protein heteromer that contains a nuclear factor kappa-B-like (NF-κB) transcription factor complex of p65 (rel-A) dimerized with p48 CCAAT enhancer- binding protein-beta (C/EBP-β) (NF-IL6α) (a p65/p48 transcription factor complex) .
As a result of this discovery, an assay to identify agents that alter (e.g., increase or decrease) the activation of the p65/p48 transcription factor complex, is now available. In one embodiment of the assay, cells comprising p65 and p48 are incubated with the agent to be tested, under conditions suitable for activation of the p65/p48 transcription factor complex. If the extent of activation of the p65/p48 transcription factor complex is greater in the presence of the agent than in the absence of the agent, the agent is said to selectively increases activation of the p65/p48 transcription factor complex. If the extent of activation of the p65/p48 transcription factor complex is less in the presence of the agent than in the absence of the agent, the agent is said to selectively decrease activation of the p65/p48 transcription factor complex. Agents identified by this assay are also contemplated by the present invention. The invention also pertains to methods of increasing activation of the p65/p48 transcription factor complex, by administering underivatized, aqueous soluble β (1-3 ) -glucan, or an agent that mimics the activity of underivatized, aqueous soluble β (1-3 ) -glucan. The present invention also relates to methods of altering (increasing or decreasing) the effect of underivatized aqueous soluble β (1-3) -glucan on cells, through modulation of the activation of the p65/p48 transcription factor complex. In one embodiment of the invention, the effect of underivatized, aqueous soluble β (1-3) -glucan on cells is altered by modulating the amount or availability of one or more components of the p65/p48 transcription factor complex, or of the complex itself, in the cell, such as by administering the p65/p48 transcription factor complex to the cells, or by administering a nucleic acid encoding p65 and p48 that is capable of being expressed in the cells.
The invention further pertains to methods of priming cells for activation by secondary stimuli, such as for activation by underivatized, aqueous soluble β (1-3) -glucan, by increasing the activation of the p65/p48 transcription factor complex. For example, the cells can be primed by increasing the availability of one or more components of the p65/p48 transcription factor complex, or of the complex itself, such as by administering the p65/p48 transcription factor complex to the cells, or by administering a nucleic acid encoding p65 and p48 that is capable of being expressed in the cells.
The present invention additionally pertains to methods of identifying the presence of immunomodulation, up- regulation of immune response, or leukocyte priming (i.e., stimulation of enhanced microbicidal activity without cytokine production) , by an agent such as an underivatized, aqueous soluble β (1-3 ) -glucan. The presence of immunomodulation, up-regulation of immune response, or leukocyte priming, by the agent is indicated by an increase in activation of the p65/p48 transcription factor complex in appropriate cells. In one embodiment, an increase in activation of the p65/p48 transcription factor complex is demonstrated by an increase in transport of the p65/p48 transcription factor complex to the nuclei of cells. In another embodiment, an increase in activation of the p65/p48 transcription factor complex is demonstrated by an increase in the production of the p65/p48 transcription factor complex.
The invention also pertains to use of the p65/p48 transcription factor complex to aid in identification of genes and gene products that contribute to or are responsible for priming of the immunomodulatory activity of agents such as underivatized, aqueous soluble β(l-3)- glucans . For example, a specific promoter sequence that activates downstream pathways that contribute to the effect of underivatized, aqueous soluble β (1-3 ) -glucan on cells can be identified by identifying binding of the p65/p48 transcription factor complex to it .
The invention further pertains to an assay for identifying agents which alter (e.g., increase or decrease) the activity of underivatized, aqueous soluble β(l-3)- glucan. The assay comprises incubating appropriate cells (e.g., cells that comprise p65 and p48) with underivatized, aqueous soluble β (1-3 ) -glucan and an agent to be tested, under conditions suitable for activation of the p65/p48 transcription factor complex. The level of activation of the p65/p48 transcription factor complex in the presence of the agent to be tested is determined, such as by measuring the level of nuclear translocation of the p65/p48 transcription factor complex or the level of p65/p48 transcription factor complex that is produced; the level of activation is compared with the level of activation of the p65/p48 transcription factor complex in the absence of the agent to be tested. A difference in the level of activation of the p65/p48 transcription factor complex indicates that the agent alters the activation of underivatized, aqueous soluble β (1-3) -glucan. An increase in the level of activation in the presence of the agent indicates that the agent increases, i.e., prolongs or enhances, activity of underivatized, aqueous soluble β(l- 3) -glucan, or is an agonist of underivatized, aqueous soluble β (1-3) -glucan. A decrease in the level of activation in the presence of the agent indicates that the agent decreases, i.e., shortens or diminishes, activity of underivatized, aqueous soluble β (1-3) -glucan, or is an antagonist of underivatized, aqueous soluble β (1-3 ) -glucan. The invention also relates to agents identified by the assays described herein, and accordingly, relates to agonists and antagonists of underivatized, aqueous soluble β (1-3) -glucan activity.
The present invention also pertains to an assay for identifying agents which have the same effect as (i.e., which mimic the activity of) underivatized, aqueous soluble β (1-3) -glucan (an "underivatized, aqueous soluble β(l-3)- glucan mimic" or "underivatized, aqueous soluble β(l-3)- glucan mimetic"), and to the agents identified by the assay. This assay comprises incubating appropriate cells (e.g., cells that comprise p65 and p48) with an agent to be tested, under conditions in which activation of the p65/p48 transcription factor complex can occur. The level of activation of the p65/p48 transcription factor complex in the presence of the agent to be tested is determined, such as by measurement of the amount of p65/p48 transcription factor complex that is translocated to the nuclei of the cells, or measurement of the amount of p65/p48 transcription factor complex produced in the cells, and compared with the level of activation of the p65/p48 transcription factor complex in the presence of underivatized, aqueous soluble β (1-3) -glucan and the absence of the agent to be tested; a level of activation of the p65/p48 transcription factor complex in the presence of the agent that is comparable to the level of activation of the p65/p48 transcription factor complex in the absence of the agent, but in the presence of underivatized, aqueous soluble β (1-3) -glucan, indicates that the agent is a mimic or mimetic of underivatized, aqueous soluble β (1-3) -glucan. The assays and methods of the present invention can be used to identify agents and drugs for use in treatment of infectious disease, inflammation, autoimmune diseases, ischemia reperfusion injury, cancer, asthma and hypersensitivity disorders. The assays and methods described herein can also be used to design agents which mimic the underivatized, aqueous soluble β (1-3) -glucan effect. For example, agents or drugs, such as, but not limited to, peptides or small organic molecules can be designed with reference to the binding site of the p65/p48 transcription factor complex. In one embodiment, such agents or drugs can be designed to mimic the activity of the p65/p48 transcription factor complex, thus obtaining a mimic of underivatized, aqueous soluble β (1-3) -glucan activity. Agents and drugs designed as mimics or mimetics; agents and drugs identified by the assays and methods; as well as the p65/p48 transcription factor complex itself, can be used in any therapeutic or prophylactic applications in which underivatized, aqueous soluble β (1-3) -glucan can be used, such as for immunomodulation, leukocyte priming, hematopoiesis, prevention and treatment of infectious disease, platelet production, peripheral blood precursor cell mobilization and wound healing.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A and IB depict cold probe competition of complex formation in LPS- or PGG-Glucan-stimulated BMC2.3 cell nuclear extracts, in an electrophoretic mobility shift assay (EMSA) , using NF-κB consensus probe (Figure 1A) or NF-IL6 consensus probe (Figure IB) in various molar excess quantities (amounts indicated are relative to the 0.5 pmol radiolabeled probe added to each reaction) . The upper horizontal arrow denotes the position of the predominate complex, while the lower arrow denotes radiolabeled free probe .
Figures 2A and 2B depict antibody competition of EMSA complex formation in LPS- or PGG-Glucan-stimulated BMC2.3 cell nuclear extracts, using NF-κB consensus probe (Figure 2A) or NF-IL6 consensus probe (Figure 2B) . Antibody designations: pre (pre- immune) ; α (C/EBP- ) ; β (C/EBP-β) ; δ (C/EBP-δ) ; p50 (κBl) ; p52 (κB2) ; p65 (rel-A) . The upper horizontal arrow denotes the position of the predominate complex, while the lower arrow denotes free probe.
Figure 3 depicts immunoprecipitation of the p65/p48 complex, using ant-p48 antibody-coated beads, from BMC2.3 nuclear extracts prepared from LPS- or PGG-Glucan- stimulated BMC2.3 cells. The arrows to the right denote immunoprecipitated p65 (rel-A) antigen (upper) or putative denatured IgG leached from the matrix (lower) . The arrows to the left indicate the positions of biotinylated markers (sizes in KDa, lane M) . The lane designated "p65 M" represents non-immunoprecipitated PGG-Glucan-treated nuclear extract marker.
Figure 4 depicts EMSA analysis of nuclear extracts from inhibitor-treated control BMC2.3 cells or LPS- or PGG- Glucan-stimulated BMC2.3 cells. The upper horizontal arrow denotes the position of the predominate complex bound to the NF-κB consensus probe, while the lower arrow denotes free probe.
Figures 5A and 5B depict the effects of inhibitors on nuclear titers of p65 and p48 in LPS- or PGG-Glucan-treated BMC2.3 cells, using anti-p65 (Figure 5A) or anti-p48 (Figure 5B) antibody. The arrows to the right denote antigens p65 or p48.
Figure 6 depicts the effects of inhibitors on I B phosphorylation in whole cell extracts from LPS- or PGG- Glucan-treated BMC2.3 cells. The arrow to the right denotes phosphorylated antigen IκB.
Figure 7 depicts antibody competition of EMSA complex formation in LPS- or PGG-Glucan-stimulated human neutrophil nuclear extracts, using NF- B consensus. Antibody designations: pre (pre- immune) ; p50 (κBl) ; p52 (κB2); p65 (rel-A) . The horizontal arrow denotes the position of the predominate complex.
Figure 8 depicts antibody competition of EMSA complex formation in LPS- or PGG-Glucan-stimulated human neutrophil nuclear extracts, using NF-κB consensus probe. Antibody designations: pre (pre- immune) ; (C/EBP-α) ; β (C/EBP-β) ; Y (C/EBP-γ) . The horizontal arrow denotes the position of the predominate complex.
Figure 9 depicts cold probe competition of EMSA complex formation in PGG-Glucan-stimulated human neutrophil cell nuclear extracts, using NF-κB consensus probe in various molar excess quantities (amounts indicated are relative to the 0.5 pmol radiolabeled probe added to each reaction) . The lower horizontal arrow denotes the position of the predominate complex, while the upper arrow denotes radiolabeled free probe.
DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to the discovery that underivatized, aqueous soluble β (1-3) -glucan (see U.S.
Serial No. 07/934,015, filed August 21, 1992, the teachings of which are incorporated herein by reference) activates a protein heterodimer (heteromer) that is a nuclear factor kappa-B-like (NF-κB) transcription factor complex containing subunit p65 (rel-A) attached to p48 CCAAT enhancer-binding protein-beta (C/EBP-β) (NF-IL6 ) . It further pertains to the discovery that underivatized, aqueous soluble β (1-3 ) -glucan increases phosphorylation of the inhibitor kappa-B-alpha (IκB-α) , and involves protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways. Underivatized, aqueous soluble β (1-3) -glucans are also described in U.S. Serial Nos. 08/400,488, 08/432,303, 08/373,251 and 08/469,233 and U.S. Patent Nos. 5,322,841, 5,488,040, 5,532,223, 5,622,939 and 5,633,369. Results of work described herein characterize this transcription factor complex activated by underivatized, aqueous soluble β (1-3) -glucan (also known as PGG-Glucan), and clearly differentiate it from previously described transcription factor complexes, while revealing important information about the mechanism of underivatized, aqueous soluble β(l- 3) -glucan biological activity.
As described in the Examples, treatment of mouse BMC2.3 macrophage cells with PGG-Glucan activates a protein heterodimer, nuclear factor kappa-B-like (NF-κB) transcription factor complex containing subunit p65 (rel-A) attached to p48 CCAAT enhancer-binding protein-beta (C/EBP- β) (NF-IL6α) (herein referred to as a "rel-A/CEBP-β p65/p48 dimer transcription factor complex" or a "p65/p48 complex") and increases its concentration in the nucleus. As used herein, "activation" of the p65/p48 transcription factor complex refers to, for example, translocation of the complex to the nucleus, formation of the complex from the separate components, increased production of the separate components or of the complex, and/or an increase in the ability of the complex to bind specific DNA sequences. Experiments were conducted in which nuclear extracts were prepared and protein/DNA complex formation assessed by electrophoretic mobility shift assay (EMSA) . Underivatized, aqueous soluble β (1-3 ) -glucan resulted in the binding of the p65/p48 complex to an NF-κB specific32P-labeled oligonucleotide probe; however, the p65/p48 complex did not interact with any rel antibody other than p65 in antibody-EMSA experiments (data not shown) , thereby indicating that the p48 subunit was not in the known rel family.
Furthermore, activation of the p65/p48 complex by PGG- Glucan did not result from potential trace amounts of LPS endotoxin contamination of the glucan preparation; the predominant LPS-activated p65/p50 complex was not detected in PGG-Glucan-treated cells (data not shown) . Because the predominant complex bound to the NF-kB EMSA consensus probe is different in LPS- versus PGG-Glucan-stimulated cells, the signaling pathways are different for these two activators in BMC2.3 cells. Immunoblots (see Figure 5 and accompanying text) indicated that the nuclear concentrations of p65 and p48 increased after treatment with PGG-Glucan or LPS. For the PGG-Glucan-treated cells, anti-C/EBP-β immunoprecipitation experiments (see Figure 3 and accompanying text) demonstrated the nuclear translocation of the p65/p48 complex, or of C/EBP-β homodimers . In contrast, for the LPS-treated cells, immunoprecipitation experiments demonstrate nuclear translocation of the classic p65/p50 complex widely known to be activated and translocated in macrophage cells by this activator (for reviews see Baldwin, A.S., Annu . Rev. Immunol . 14:649-683 (1996); Sweet, M.J. and D.A. Hume, J. Leukocyte Biol . 60 : 8 - 26 (1996); Barnes, P.J. and M. Karin, N. Engl . J. Med . 336:1066-1071 (1997)). In addition, the immunoblot experiments indicated that the p65 and p48 antigens appeared to be attached together in the nuclei of the cells stimulated with PGG-Glucan, and the titer of this complex increased in the nucleus after PGG-Glucan treatment . Experiments using Calphostin-C (CAL) , a highly specific inhibitor of the PKC family of proteins, with little effect on other kinases (see, e.g., Tamaoki, T. and H. Νakano, Bio/Technology 8:732-735 (1990), or using Genistein (GEΝ) , a highly specific inhibitor of tyrosine kinases, with little effect for serine- and threonine- specific protein kinases, including PKC (Akiyana et al . , 1987; Platanias, L.C. and O.R. Colamonici, J. Biol . Chem . 267:24053-24057 (1992)), demonstrated that CAL or GEΝ prevented formation of the predominant ΝF-κB EMSA complex (see Figure 4 and accompanying text) . These results implicated both PKC and PTK pathways in PGG-Glucan- activated cells.
The data also indicate that IκB- may be involved in PGG-Glucan signal transduction. Because the phosphorylation of IκB- stimulated by PGG-Glucan was blocked by CAL (see Figure 6 and accompanying text) , the data support the idea that PKC is part of the signalling pathway leading to the IκB-α phosphorylation (Ghosh, S. and D. Baltimore, Nature 344:678-682 (1990); Diaz-Meco, M. et al . , EMBO J. 13:2842-2848 (1994)). However whether IκB- is directly phosphorylated by PKC isozymes is still unclear (Janosch, P. et al . , J. Biol . Chem . 271:13868-13874 (1996) ) .
Thus, the data described herein are consistent with a model for PGG-Glucan activation of BMC2.3 cells whereby activation of PKC and/or PTK pathways leads to phosphorylation of IκB- , the release and translocation to the nucleus of p65/p48, and increase in p65/p48 DΝA-binding activity. Applicants have, for the first time, demonstrated the activation of a p65/p48 complex by soluble β-glucans. Complexes containing p65 and C/EBP-β have not previously been identified in vivo, although in vi tro transfection studies have shown a general interaction between all members of the ΝF-κB and C/EBP families (Stein, B. et al . , Mol . Cell Biol. 13:3964-3974 (1993)). A complex containing p66 (believed to be rel-A) , C/EBP-δ, and at least 3 other unidentified proteins has been isolated from the avian T-cell line MSB-1 (Diehl, J.A. and M. Hannink, Mol . Cell Biol . 14:6635-6646 (1994)). A complex containing p65 and C/EBP-δ has been isolated from the liver tissue of LPS-treated rabbits, however the composition of the complex is unknown, possibly containing a multimer of the heteromer (Ray, A. et al . , J". Biol . Chem . 270 : 1365- 1314 (1995)).
The p65/p48 transcription factor complex can be used to aid in identification of genes and gene products that contribute to or are responsible for priming of the immunomodulatory activity of underivatized, aqueous soluble β (1-3) -glucans . For example, the p65/p48 transcription factor complex can be used to bind to a specific promoter sequence to activate downstream pathways that contribute to the effect of underivatized, aqueous soluble β (1-3 ) -glucan on cells. Identification of promoters to which the p65/p48 transcription factor complex binds can help identify the genes and/or gene products that are affected by activation of the p65/p48 transcription factor complex. Genes and/or gene products that are thereby identified can be targeted (e.g., for activation or for suppression) to achieve an effect similar to that of underivatized, aqueous soluble β (1-3) -glucan. Agents which alter (increase or decrease) activation of the p65/p48 transcription factor complex can be identified by an assay in which the level of activation of the p65/p48 transcription factor complex in the presence of the agent is compared with the level of activation of the p65/p48 transcription factor complex in the absence of the agent. Appropriate cells (e.g., cells that comprise p65 and p48) are incubated with the agent to be tested, under conditions suitable for activation of p65/p48 transcription factor complex. Activation is measured, for example, by measuring the amount of p65/p48 transcription factor complex that is translocated to the nuclei of the cells, or the amount of p65/p48 transcription factor complex that is produced in the cells. If the level of activation of the p65/p48 transcription factor complex in the presence of the agent is greater than the level of activation of the p65/p48 transcription factor complex cells maintained under the same conditions but in the absence of the agent, then the agent is an agent that increases activation of the p65/p48 transcription factor complex. If the level of activation of the p65/p48 transcription factor complex in the presence of the agent is less than the level of activation of the p65/p48 transcription factor complex cells maintained under the same conditions but in the absence of the agent, then the agent is an agent that decreases activation of the p65/p48 transcription factor complex. Agents identified by this assay are within the scope of the present invention. In one embodiment, the activation of p65/p48 transcription factor complex can be increased by administration of underivatized, aqueous soluble β (1-3) -glucan, or a mimic of underivatized, aqueous soluble β (1-3) -glucan, as described below.
The effect of underivatized, aqueous soluble β(l-3)- glucan on cells can be altered (increased or decreased) by modulating activation of the p65/p48 transcription factor complex. For example, the level of activation of the p65/p48 transcription factor complex in cells can be altered through use of an agent that alters the amount of p65 and/or p48, or the amount of the p65/p48 transcription factor complex, that is present in cells (e.g., an agent that increases or decreases transcription or translation of p65 and/or p48, or that increases or decreases the activity of p65 and/or p48, or increases or decreases availability or p65 and/or p48 in cells, such as by releasing p65 and/or 48 from other binding partners, or by binding to p65, to p48, or to the p65/p48 transcription factor complex) . In a preferred embodiment, the effect of underivatized, aqueous soluble β (1-3) -glucan on cells is altered by an agent that increases the amount of p65/p48 transcription factor complex that is available for activation (e.g., use of nucleic acid encoding p65 and p48 proteins, to express the components of the p65/p48 transcription factor complex) . The cells are contacted with the agent in an appropriate manner to achieve the desired effect . In another preferred embodiment, the effect of underivatized, aqueous soluble β (1-3) -glucan on cells is altered by an agent that decreases the availability of p65 or p48, such as an antibody to p65 or to p48.
The presence of the immunomodulation, upregulation of immune response, or leukocyte priming, that is caused by an underivatized, aqueous soluble β (1-3) -glucan can be monitored by monitoring the activation of the p65/p48 transcription factor complex, such as by monitoring translocation of the p65/p48 transcription factor complex to the nucleus, or monitoring the production of the p65/p48 transcription factor complex. The presence of immunomodulation, upregulation of immune response, or leukocyte priming, is indicated by an increase in activation of the p65/p48 transcription factor complex in appropriate cells (e.g., cells that comprise p65 and p48, such as macrophage cells, peripheral mononuclear cells, monocytes, or BCM2.3 cells) .
The invention further pertains to an assay for identifying agents which alter the activity of underivatized, aqueous soluble β (1-3) -glucan. This assay comprises incubating appropriate cells (e.g., cells comprising p65 and p48, such as macrophage cells, peripheral mononuclear cells, monocytes, PMN or BCM2.3 cells) with underivatized, aqueous soluble β (1-3) -glucan and an agent to be tested under conditions that are suitable for activation of the p65/p48 transcription factor complex (e.g., conditions suitable for binding of underivatized, aqueous soluble β (1-3) -glucan to the receptor for underivatized, aqueous soluble β (1-3 ) -glucan) . The extent of activation of the p65/p48 transcription factor complex in the presence of the agent to be tested is determined (e.g., using radiolabeled DNA oligonucleotides specific for the p65/p48 transcription factor complex, as described in the Examples) , and compared with the extent of activation of the p65/p48 transcription factor complex in the same type of cells, under comparable conditions but in the absence of the agent to be tested. A difference in the extent of activation indicates that the agent alters the activity of underivatized, aqueous soluble β (1-3) -glucan. An increase in the activation of the p65/p48 transcription factor complex in the presence of the agent indicates that the agent increases (i.e., prolongs or enhances) the activity of underivatized, aqueous soluble β (1-3 ) -glucan. A decrease in the activation of the p65/p48 transcription factor complex in the presence of the agent indicates that the agent decreases (i.e., shortens or diminishes) the activity of underivatized, aqueous soluble β (1-3 ) -glucan.
An assay for identifying agents which mimic (e.g., have approximately the same activity as) underivatized, aqueous soluble β (1-3 ) -glucan (for example, an agent which activates the p65/p48 transcription factor complex) can be used to identify an underivatized, aqueous soluble β(l-3)- glucan mimic. For the assay, appropriate cells (e.g., cells that comprise p65 and p48) are used. A sample of cells is incubated with an agent to be tested (the "test agent") under conditions in which activation of the p65/p48 transcription factor complex can occur. Subsequently, the level of activation of the p65/p48 transcription factor complex, if any, in the presence of the agent to be tested is determined by appropriate methods, such as by measurement of the amount of p65/p48 transcription factor complex that is translocated to the nuclei of the cells. This level of activation (the "test level") is compared with a base level (absence of the agent and β (1-3 ) -glucan) or a control level, which is the level of activation of the p65/p48 transcription factor complex in a sample of the same type of cells maintained under the same conditions, but in the absence of the agent to be tested and in the presence of underivatized, aqueous soluble β (1-3 ) -glucan. If the test level is comparable to the control level (i.e., the activation of the p65/p48 transcription factor complex is similar) , or if the test level is greater than the control level, then the agent increases activation of the p65/p48 transcription factor complex, and therefore is a mimic of aqueous soluble β (1-3 ) -glucan. Agents identified by this assay as being mimics of aqueous soluble β(l-3)- glucan are also within the scope of the invention. Agents or drugs, such as, but not limited to, peptides, CH3 polymers, lipids, nucleic acids or small organic molecules can be designed as mimics of aqueous soluble β (1-3 ) -glucan, with reference to the binding site of the p65/p48 transcription factor complex. In one embodiment, such agents or drugs can be designed to mimic the activity of the p65/p48 transcription factor complex, thus obtaining a mimic of underivatized, aqueous soluble β (1-3 ) -glucan activity. These agents and drugs designed as mimics; agents and drugs identified by the assays and methods described above; and/or the p65/p48 transcription factor complex itself, can be used in any therapeutic or prophylactic application in which underivatized, aqueous soluble β (1-3) -glucan can be used. Mimetics and mimics have been described in U.S. Serial No. 08/902,586.
The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are incorporated herein by reference.
EXAMPLE 1 MATERIALS AND METHODS Materials PGG-Glucan (BETAFECTIN™) [poly- (1-6) -β-D- glucopyranosyl- (1-3) -β-D-glucopyranose] manufactured by Alpha-Beta Technology, Inc. (Worcester, MA) was determined to be endotoxin-free by a Limulus amebocyte lysate assay (LAL assay; QCL-1000; Whittaker Bioproducts, Inc.). E. coli lipopolysaccharide (LPS) was purchased from Sigma Chemical Company (St. Louis, MO). Protease inhibitors (phenylmethylsulfonyl fluoride (PMSF) , chymostatin, pep- statin-A, aprotinin, antipain, and leupeptin) and other routine chemicals were purchased from Sigma or Boehringer Mannheim (Indianapolis, IN) . Calphostin-C (CAL) and Genistein (GEN; 4 ' , 5 , 7-Trihydroxyisoflavone) were purchased from Calbiochem. DNA oligomers were custom synthesized (Genosys Biotechnologies, Inc., Woodlands, TX) . All antibodies to transcription factor proteins were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) , all are polyclonal IgG induced against synthetic peptides with cross-reactivity for mouse: p50 (kBl, goat anti-mouse, #SC-1192X) , p52 (kB2, rabbit anti-mouse, #SC-298X) , p65 (rel-A, rabbit anti-human, #SC-372X) , p68 (rel-B, rabbit anti-mouse, #SC-226X) , p75 (C-rel, rabbit anti-human, #SC- 272X) , C/EBP- (p42 , rabbit anti-rat, #SC-61X) , C/EBP-β (NF-IL6a, rabbit anti-rat, #SC-150X) , C/EBP-δ (NF-IL6b, rabbit anti-rat, #SC-151X) . Phospho-specific IκB- (Ser32) polyclonal IgG (#9241, New England Biolabs, Beverly, MA) was induced in rabbits against a synthetic phospho-Ser32 peptide .
Cell Stimulations
Murine bone marrow-derived macrophage cells (BMC2.3 ; kindly provided by K.L. Rock, University of Massachusetts Medical School, Worcester, MA) were cultured in DMEM supplemented with 10% heat-inactivated FCS, 1 mM Glutamine, and 1% Penn/Strep (all Life Technologies, Gaithersburg, MD) . LPS or PGG-Glucan were added to the media at final concentrations of 1 μg/ml and 3 μg/ml, respectively, and 37°C incubations continued for 0.5-1.0 hr prior to preparation of nuclear extracts. During- inhibitor experiments, Calphostin-C (250 nM) or Genistein (15 μM) were added to the medium 5 min prior to stimulation.
Nuclear Extractions for Electrophoretic Mobility Shift Assays
Nuclear extracts for EMSAs were prepared essentially as described previously [Dignam, J.D. et al., Nucl . Acids Res . 11:1475-1489 (1983); Prywes, R. and R.G. Roeder, Cell 47:777-784 (1986)] using 1.0-2.0 x 107 cells per sample. All buffers were freshly supplemented with (final concentrations indicated in parentheses) : DTT (0.5 mM) , protease inhibitors PMSF (0.5 mM) , chymostatin, pepstatin- A, aprotinin, antipain, and leupeptin (each at 1 μg/ml) , and phosphatase inhibitors NaF (10 mM) , ZnCl2 (1 mM) , sodium orthovanadate (1 mM) , and sodium pyrophosphate (5 mM) . Aliquots of the final dialyzates were stored at -80°C and discarded after single use. Protein concentrations varied from 0.5-2.0 mg/ml as determined by a modified
Coomassie protein assay (Pierce, Rockland, IL) against a BSA standard.
Nuclear Extractions for Western Blots Nuclear extracts for Western blots were prepared essentially as described (Min, W. et al . , J. Immunol . 156:3174-3183 (1996)). Cells (1.0-2.0 x 107) were harvested at 4°C into PBS, then resuspended in 5 ml of hypotonic buffer A (10 mM HEPES pH 7.9 , 10 mM KCl, 1.5 mM MgCl2) freshly supplemented with DTT and the protease/ phosphatase inhibitors described above. Cells were incubated 15 min on ice to allow swelling and penetration of protease inhibitors, then lysed by addition of Nonidet P-40 to 0.5% (v/v) and gentle inversion. Nuclei were pelleted (1500 x g, 5 min, 4°C) and resuspended in 250 ml of extraction buffer (20 mM HEPES pH 7.9 , 0.45 M NaCI, 1 mM EDTA) freshly supplemented with inhibitors as above . Nuclear suspensions were incubated for at least 30 min at 4°C with intermittent agitation, then centrifuged (14000 x g, 5 min, 4°C) to pellet nuclear debris. Supernatants were mixed with an equal volume of dilution buffer (20 mM HEPES pH 7.9, 0.1 M KCl, 0.2 mM EDTA, 20% glycerol) . Aliquots were stored at -80°C and discarded after single use. Protein concentrations varied from 1-4 mg/ l .
Whole Cell Extracts for Western Blots Whole cell extracts were used for phospho-I B- immunoblots. BMC2.3 cells (1.0-2.0 x 107 per sample) were washed in IX PBS, 20 mM EDTA, then resuspended in 250 μl of fresh lysis buffer (20 mM HEPES pH 7.9, 10 mM KCl, 300 mM NaCI, 1 mM MgCl2, 0.1% Triton-X 100, 20% glycerol, 0.5 M DTT, freshly supplemented with inhibitors as described above. Suspensions were incubated for at least 10 min on ice to lyse the cells, then centrifuged (14000 x g, 5 min, 4°C) to pellet cell debris. Supernatant aliquots were stored at -80°C and discarded after single use. Protein concentrations varied from 2-6 mg/ml. Electrophoretic Mobility Shift Assays
Transcription factor activations were assayed using an electrophoretic mobility shift assay (EMSA) as described previously (for review see Kerr, L.D., Methods in Enzymology 254:619-632 (1995)). NF-kB consensus synthetic duplex probe (Lenardo, M.J. and D. Baltimore, Cell 58 : 221 - 229 (1989)) was 5 'AGTTGAGGGGACTTTCCCAGGC [SEQ ID NO:l], NF-IL6 consensus synthetic duplex probe (Mahoney, C.W. et al . , J. Biol . Chem . 267:19396-19403 (1992)) was 5 ' TGCAGATTGCGCAATCTGCA [SEQ ID NO: 2]. Duplex probes were end-labeled with32P using polynucleotide kinase and [γ-32P]ATP. Labeled probe (0.5 pmol) was mixed with 3 μg of nuclear extract protein in a solution containing 10 mM Tris-HCl pH 7.5, 50 mM NaCI , 1 mM EDTA, 1 mM DTT, 5% glycerol, 0.02% β-mercaptoethanol, 0.1-1.0 mg of poly(dl/dC) (Pharmacia) . Reactions were incubated at 25°C for 20 min to allow complex formation, then electrophoresed under non-denaturing conditions through 4% polyacrylamide gels in 0.5X TBE buffer (45 mM Trisma base, 45 mM boric acid, 1 mM EDTA) . Gels were dried onto 3MM paper. Bands were visualized by autoradiography at -80°C with one intensifying screen, and quantitated by laser densitometry.
EMSA Probe Competitions
Non-radiolabeled "cold" duplex NF-kB or NF-IL6 oligomers (see above) , or "cold" mutant duplex oligomers [NF-kB 5 ' AGTTGAGGCGACTTTCCCAGGC [SEQ ID NO : 3 ] (Lenardo, M.J. and D. Baltimore, Cell 58:227-229 (1989)); NF-IL6 TGCAGAGACTAGTCTCTGCA [SEQ ID NO:4] (Mahoney, C.W. et al . , J". Biol . Chem . 267:19396-19403 (1992))] were added to the EMSA reaction for 20 min at 25°C prior to32P-probe addition. Following radiolabeled probe addition, the incubation was continued at 25°C for an additional 20 min prior to electrophoresis.
EMSA Antibody Competitions Antibody to a specific transcription factor protein (see Materials) (3 μg) was added to a 25°C EMSA reaction for 60 min prior to32P-probe addition. Following labeled probe addition, incubations were continued at 25°C for an additional 20 min prior to electrophoresis.
Western Blots
Western blots were performed essentially as described (Adams, D.S. et al . , J. Biol . Chem . 266:8476-8482 (1991)) using 5 μg of nuclear extract protein per lane for p65, 10 μg for p48, and 20 μg for phospho-IκB-α . Primary antibody incubations were for 2 hr at 25°C in fresh blocking buffer (IX PBS, 1% casein, and 0.2% Tween-20) containing rabbit anti-human p65 (#SC-372X, 0.1 mg/ml), rabbit anti-rat p48 (#SC-150X, 0.2 mg/ml), or rabbit anti-human phospho-IκB-α (#9241, 1:1000). Membranes were washed twice m PBS-Tween (IX PBS, 0.05% Tween-20) , and then incubated with the secondary antibody. Secondary antibody incubations were for 2 hr at 25 °C in fresh blocking buffer containing goat anti -rabbit -HRP IgG (Pierce #31460, 0.4 μg/ml). Detection of biotinylated marker proteins (Biorad, Broad Range) used streptavidin-HRP (Pierce #21126, 0.5 μg/ml).
Chemiluminescent detection of HRP used luminol/H202 (Pierce) . Exposure for autoradiography was at 25°C for 1-5 min. Preparative Immunoprecipitations
Anti-C/EBP-β (see Materials) (100 μg) was linked to 500 μl of CARBOLINK™ gel (Pierce) via the oxidized carbohydrate IgG moiety using the manufacturer's procedure. Approximately 80% of the IgG coupled to the resin (0.16 μg IgG/μl resin). Resin (20 μl of 50% slurry in PBS, 1.6 μg IgG) was added directly to nuclear extracts (200 μg) and the samples were incubated overnight at 4°C with constant shaking. Beads were washed 3X in PBS, and proteins eluted in protein electrophoresis sample buffer by boiling 2 min. Samples were analyzed on immunoblots using p65 IgG as primary antibody.
EXAMPLE 2 IDENTIFICATION OF PROTEIN COMPONENTS OF FACTORS ACTIVATED BY PGG-GLUCAN In an attempt to identify specific proteins associated with the predominant PGG-Glucan-activated EMSA complexes, antibody-electrophoretic mobility shift assays (EMSA) experiments were performed as described above. LPS was used as a positive control because it is well documented that in macrophage cells LPS activates p65/p50 heterodimer NF-KB complexes, and C/EBP-β/C/EBP-β homodimer NF-IL6 complexes (for reviews see Baldwin, A.s., Annu . Rev. Immunol . 14:649-683 (1996); Sweet, M.J. and D.A. Hume, J. Leukocyte Biol . 60 : 8 -26 (1996); and Barnes, P.J. and M. Karin, N. Engl . J. Med . 336 : 1066 - 1011 (1997)) .
Results of these experiments (data not shown) indicated that, as expected for LPS-stimulated extracts, complex formation with the ΝF-κB probe was prevented by anti-p65 (rel-A) and anti-p50 (κBl) , but was not prevented by incubation with pre-immune serum or anti-p52 (κB2) . In contrast for the PGG-Glucan-stimulated extracts, anti-p65 prevented NF-κB-like complex formation, but anti-p50 or anti-p52 did not. Commercially available antibodies to other rel family members did not compete for either complex formation (data not shown) . In neither case (LPS or PGG- Glucan) were the antibodies found to produce supershifted complexes. The LPS-stimulated complex had approximately the same size as the complex activated by PGG-Glucan.
These data demonstrate that PGG-Glucan treatment of mouse BMC2.3 macrophage cells activates an NF-κB-like transcription factor complex containing subunit p65 (rel-A) attached to an unidentified cohort with a size approximately 50 kDa, and also activates an NF-IL6-like complex containing C/EBP-β (NF-IL6α) attached to an unidentified cohort with a size similar to C/EBP-β. Because formation of the NF-κB/oligo probe complex was not blocked by antibodies against p50 (κBl) , p52 (κB2) , p68 (rel-B) , or p75 (C-rel) , the unknown cohort was presumed to be a non-rel family member. In probe competition EMSAs, unlabeled wild-type probe strongly competed with labeled probe for its corresponding complex, while mutant probe did not (data not shown) . Thus the formed complexes appeared to represent highly specific interactions.
To investigate more thoroughly the lack of interaction of p50 antibody with the PGG-Glucan-stimulated NF-κB-like complex, Western blots were performed to measure the nuclear levels of p65 and p50 antigens (data not shown) . Levels of p65 antigen increased in the nucleus after treatment of the cells with either LPS or PGG-Glucan, while only LPS increased the nuclear levels of p50. The Western data thus indicate that treatment of the BMC2.3 cells with PGG-Glucan increases the nuclear titer of p65, probably by translocation from the cytoplasm (as is well documented for LPS-induced NF-κB) . The p50 antibody-EMSA and Western data together suggest that the PGG-Glucan stimulated NF-κB-like complex does not include a p50 subunit.
For the NF-IL6 antibody, as expected for LPS- stimulated extracts, complex formation with the NF-IL6 probe was prevented by anti-C/EBP-β, but was not prevented by incubation with pre-immune serum or anti-C/EBP- or anti-C/EBP-δ (data not shown) . Anti-C/EBP-γ was not tested because it was not commercially available. Similar data was obtained with the PGG-Glucan stimulated extracts. As was the case for the NF-κB-like complexes, neither NF-IL6- like complex (LPS or PGG-Glucan) was supershifted with these antibodies. These data demonstrate that the PGG- Glucan stimulated NF-IL6-like complex includes C/EBP-β as at least one of its subunits . Because the LPS-stimulated complex has approximately the same size as the complex activated by PGG-Glucan, it suggests that the two complexes are identical homodimers , or the unidentified cohort has a size approximately equal to that of C/EBP-β.
EXAMPLE 3 PGG-GLUCAN ACTIVATES A p65/p48 Rel/bZIP COMPLEX Experiments were performed to test the ability of the NF-IL6 consensus probe to compete for protein complex formation with a labeled NF-κB consensus probe. BMC2.3 cells were stimulated with either LPS (1 μg/ml) or PGG- Glucan (3 μg/ml) for 60 min. EMSAs were performed using NF-KB consensus probe, or NF-IL6 consensus probe, as described in Example 1, except various molar excess quantities of non-radiolabeled "cold" oligomer or "cold" mutant oligomer were added to the LPS- or PGG-Glucan- stimulated BMC2.3 nuclear extracts prior to32P-probe addition. As indicated above, LPS-treated BMC2.3 cells were used as positive controls since treatment of macrophage cells with this agent is well known to activate classic NF-KB (p65/p50 rel/rel) and NF-IL6 (C/EBP-β- homodimer) complexes (for reviews see Baldwin, A.S., Annu . Rev. Immunol . 14:649-683 (1996); Sweet, M.J. and D.A. Hume, J". Leukocyte Biol . 60 : 8 -26 (1996); Barnes, P.J. and M. Karin, N. Engl . J. Med . 336 : 1066 - 1011 (1997)) . Indeed, previous data (not shown) indicated that these were the predominant complexes bound to the consensus probes following LPS treatment.
Results are shown in Figures 1A (ΝF-κB consensus probe) and IB (NF-IL6 consensus probe) . A single predominate DNA/protein complex was formed with each probe. Although higher-order complexes were occasionally observed, their formation was very weak and inconsistent. As expected for LPS-treated cells, cold NF-IL6 consensus probe did not compete for NF-κB complex formation (even at 50- fold molar excess relative to labeled probe (Figure 1A) and cold NF-KB probe did not compete for NF-IL6 complex formation (Figure IB) . However for PGG-Glucan-treated cells, cold NF-IL6 probe strongly competed for NF-κB complex formation, while cold mutant NF-IL6 probe did not (Figure 1A) . Cold NF-κB probe did not compete for NF-IL6 complex formation (Figure IB) . These data indicate that the predominant complex bound to the NF-κB consensus probe may contain a member of the NF-IL6 family of proteins. This complex appears to be different than the predominant complex bound to a NF-IL6 consensus probe which probably does not contain a rel family member.
Antibody-competition EMSAs designed to determine the specific proteins associated with the predominant EMSA complexes were also performed. All antibodies used for these experiments had cross-reactivity for the appropriate mouse protein (see Materials, Example 1) . EMSAs were performed using NF-κB consensus probe or NF-IL6 consensus probe as described in Example 1, except that 3 μg of competing antibody was added to a 3 μg aliquot of LPS- (1 μg/ml, 60 min) or PGG-Glucan- (3 μg/ml, 60 min) stimulated nuclear extract prior to32P-probe addition.
The results are shown in Figures 2A (NF-κB consensus probe) and 2B (NF-IL6 consensus probe) . For the LPS- stimulated extracts, neither pre- immune serum nor any of the NF-IL6 antibodies tested (C/EBP- , β, δ) competed for NF-κB complex formation. C/EBP-γ antibody was not tested because it was not commercially available. In contrast, for PGG-Glucan-stimulated "extracts, anti-C/EBP-β strongly competed for NF-κB complex formation. The anti-C/EBP-β block did not produce any supershifted complexes, in agreement with a previous study (data not shown) and other labs using similar antibodies from the same commercial source (Lewis, J. et al . , Mol . Cell Biol . 14:5701-5709 (1994) ) or similar antibodies from different sources (Ray, A. and B. Ray, DNA Cell Biol . 14:795-802 (1995); Petersen, U.M. et al . , EMBO J. 14:3146-3158 (1995)). For both the LPS and PGG-Glucan stimulations, none of the rel antibodies tested [p50 (κBl) , p52 (κB2), p65 (rel-A)] competed for NF- IL6 complex formation (Figure 2B) . Other commercially available antibodies to rel family members were also tested (data not shown) including rel-B (p68) and C-rel (p75) , and they too did not compete for NF-IL6 complex formation. These data demonstrate that the NF-κB-like complex (previously shown to include p65) also includes C/EBP-β as one of its subunits. The NF-IL6-like complex does not appear to contain a rel member.
To investigate more thoroughly the apparent existence of a p65/CEBP-β complex activated by PGG-Glucan, preparative immunoprecipitation experiments were performed using anti-C/EBP-β attached to CARBOLINK™ beads. Nuclear extracts prepared from BMC2.3 cells stimulated with LPS (1 μg/ml, 60 min) or PGG-Glucan (3 μg/ml, 60 min) were incubated with C/EBP-β-beads , and the bound material analyzed by anti-p65 immunoblots as described in Example 1. Results are shown in Figure 3. Lanes designated "PGG + Cal" and "PGG + Gen" contain samples that were pre- treated with Calphostin-C or Genistein, respectively, for 5 minutes prior to addition of PGG-Glucan activator. For untreated extracts, or those treated with LPS, no p65 antigen appeared to be isolated by the immunoprecipitation. However, extracts stimulated with PGG-Glucan showed a clear p65 band. All three treatments showed a strong band approximately 50-55 kDa in size that presumably represents IgG denatured from the immunoprecipitation gel via the elution technique (boiling in β-mercaptoethanol -containing sample buffer) and detected by the anti -rabbit secondary antibody, but this strong band did not obscure the p65 antigen. The opposite experiment, a p65- immunoprecipitation followed by an anti-C/EBP-β immunoblot, was attempted; however, in this case the 50-55 kDa putative IgG band obscured the C/EBP-β antigen (which in these BMC2.3 cells is approximately 48 kDa, see Figure 5 and accompanying discussion below) even when more gentle elution procedures with acidic-glycine were performed. These data demonstrate that the p65 (rel-A) and p48 (C/EBP- β) antigens appear to be attached together in BMC2.3 nuclei stimulated with PGG-Glucan, and the titer of this complex increases in the nucleus after PGG-Glucan treatment. Hereafter, for sake of abbreviation, this complex is referred to as p65/p48.
EXAMPLE 4 POTENTIAL INVOLVEMENT OF PKC AND TYROSINE KINASES In order to investigate which kinase pathways might be involved in the activation of the p65/p48 complex, inhibition experiments were performed. Because much more is known about the activation of rel factors in macrophage cells than NF-IL6, all subsequent experiments focused on the activation of the p65/p48 complex.
Calphostin-C (CAL) is a cell -permeable highly specific inhibitor of the PKC family of proteins that interacts with this family's unique regulatory domain, not the catalytic domain common to other kinases (for a review see Tamaoki , T. and H. Nakano, Bio /Technology 8 : 132 - 135 (1990)). CAL has a 50% inhibition concentration (IC50) of 50 nM for PKC, 50 μM for cAMP-dependent protein kinases, 28 μM for cGMP- dependent protein kinases, 5 μM for myosin light -chain kinase, and 50 μM for p60 protein tyrosine kinase (Ibid.) . Genistein (GEN) is a highly specific inhibitor of tyrosine kinases (IC50 2.6 μM) with little effect for serine- and threonine-specific protein kinases, including PKC (Akiyana et al . , 1987; Platanias, L.C. and O.R. Colamonici, J. Biol . Chem . 267:24053-24057 (1992)).
NF-KB EMSAs for BMC2.3 cells treated with CAL or GEN at 5X IC50 concentrations for 5 min prior to activation with LPS or PGG-Glucan were performed. Nuclear extracts were prepared from untreated BMC2.3 cells (Control), or from LPS- (1 μg/ml, 60 min) or PGG-Glucan (3 μg/ml, 60 min) treated cells, as described in Example 1. LPS was used for comparison here since this stimulator is known to utilize a phorbol -insensitive PKC-zeta (PKC-ζ) isoform to activate NF-KB in macrophage cells (Lozano, J. et al . , J. Biol . Chem . 269 : 19200 - 19202 (1994)), and GEN has been shown to block the LPS-induced activation of NF-κB and increase in cytokine production (IL-lβ, IL-6, TNF-α) in human monocytes (Geng, Y. et al . , J. Immunol . 151:6692-6700 (1993)).
Results are shown in Figure 4. Lanes designated "Cal" or "Gen" represent extracts prepared from cells pre-treated with Calphostin-C or Genistein, respectively, for 5 minutes prior to activation. As expected, treatment of BMC2.3 cells with either CAL or GEN prior to LPS activation prevented formation of the predominant NF-κB EMSA complex. Similar results were obtained with PGG-Glucan-stimulated cells. Neither inhibitor was found to lower cell viability (viability remained >90%) thus the blocked EMSA signals did not result from cell death. These data demonstrate that PGG-Glucan activation of the p65/p48 complex appears to involve PKC and PTK pathways .
Previous experiments (data not shown) demonstrated that the nuclear concentration of p65 antigen (but not p50) increased in PGG-Glucan-treated BMC2.3 cells, probably by translocation from the cytoplasm. In order to determine whether CAL or GEN could inhibit this apparent nuclear translocation event, p65 and p48 immunoblots were performed. BMC2.3 nuclear extracts prepared from LPS- (1 μg/ml, 60 min) or PGG-Glucan (3 μg/ml, 60 min) treated cells were analyzed by anti-p65 or anti-p48 immunoblots, as described in Example 1.
Results are shown in Figures 5A (anti-p65) and 5B (anti-p48) . Lanes designated "Cal" or "Gen" represent extracts prepared from cells pre-treated with Calphostin-C or Genistein, respectively, for 5 minutes prior to activation. As in the previous experiments, the nuclear titer of p65 antigen increased in LPS- or PGG-Glucan- treated cells (Figure 5A) . This p65 increase was blocked by pre-treatment of the cells with either CAL or GEN. Similar results were obtained with C/EBP-β (Figure 5B) . The C/EBP- β antigen in these BMC2.3 cells appears to have a molecular weight of approximately 48 kDa. This size is close to that of p50, and may help explain why the LPS-activated p65/p50 complex appears to have the same mobility on non-denaturing gels as the PGG-Glucan-activated p65/p48 complex. For the p65 antigen, the LPS treatment is probably translocating the p65/p50 complex and the PGG-Glucan treatment is probably translocating the p65/p48 complex. For the p48 antigen, the LPS treatment is probably translocating the C/EBP-β homodimer complex. These data do not indicate whether the p48 immunoblot for PGG-Glucan treated cells is monitoring the apparent translocation of a C/EBP-β homodimer or the p65/p48 complex. In order to determine whether CAL or GEN can block the appearance of p65/p48 in the nucleus, immunoprecipitation experiments were performed, as described above. Results, shown in Figure 3, demonstrate that no p65 antigen was observed in C/EBP-β-immunoprecipitated nuclear extracts pre-treated with CAL or GEN prior to PGG-Glucan activation. Taken together, the inhibition data implicate the involvement of PKC and PTK pathways in the activation and apparent nuclear translocation of p65/p48.
EXAMPLE 5 POTENTIAL INVOLVEMENT OF IκB-α In resting cells, NF-κB transcription factor dimers are present in the cytosol in an inactive state, complexed with IKB proteins, most frequently IκB-α (for reviews see Baldwin, A.S., Annu. Rev. Immunol . 14:649-683 (1996); Sweet, M.J. and D.A. Hume, J". Leukocyte Biol . 60 : 8 -26 (1996); Barnes, P.J. and M. Karin, N. Engl . J. Med .
336 : 1066 - 1011 (1997)). Activation of this trimer complex occurs via phosphorylation of IκB- at serines 32 and 36, leading to release and nuclear translocation of active ΝF- KB . Because phosphorylation of IκB at Ser-32 is essential for release of the active ΝF-κB, phosphorylation at this site is an excellent marker of NF-κB activity. In order to determine whether the PGG-Glucan activation of BMC2.3 cells increases the phosphorylation state of IκB, immunoblot experiments were performed with phospho-specific IκB-α antibody. BMC2.3 cells were treated with either LPS (1 μg/ml, 30 min) or PGG-Glucan (3 μg/ml, 30 min) (without inhibitor) , and analyzed by anti-phospho- IκB-α (Ser-32) immunoblot as described in Example 1.
The results are shown in Figure 6. Lanes designated "Cal" or "Gen" represent extracts prepared from cells pre- treated with Calphostin-C or Genistein, respectively, for 5 minutes prior to activation. These results indicate that cells treated with either LPS or PGG-Glucan without inhibitor displayed a very strong band at approximately 45 kDa relative to untreated cells. Although fainter lower MW bands were also seen in the activated lanes, we assume the strong 45 kDa band represents IκB-α because its size is similar to that of IκB-cx in other cells (approximately 40- 42 kDa, 317 aa, see above reviews) . The appearance of this band in LPS- or PGG-Glucan-stimulated cells was completely blocked by pre-incubating the cells with either CAL or GEN. These data demonstrate that phosphorylation of IκB-α increases in PGG-Glucan-treated cells, and that this phosphorylation event appears to involve PKC and PTK pathways. Because these kinase inhibitors appear to block all three types of activation events monitored here (IκB-α phosphorylation, p65/p48 DNA-binding activity in EMSAs, and apparent p65/p48 translocation) , these events may be related. EXAMPLE 6 ACTIVATION OF P65/P48 TRANSCRIPTION FACTOR COMPLEX BY PGG-GLUCAN Experiments were performed to investigate whether the p65/p48 transcription factor complex was activated in human neutrophils obtained from volunteers. Antibody- electrophoretic mobility shift assays (EMSA) experiments were performed as described above in Examples 1 and 2 ; as before, LPS was used as a positive control. Results of these experiments, shown in Figure 7, indicated that, as expected for LPS-stimulated extracts, complex formation with the NF-KB probe was prevented by anti-p65 (rel-A) and anti-p50 (κBl) , but was not prevented by incubation with pre-immune serum (pre) or anti-p52 (κB2) . In contrast for the PGG-Glucan-stimulated extracts, anti-p65 prevented NF- κB-like complex formation, but anti-p50 or anti-p52 did not. Furthermore, in experiments performed as described above, binding of a PGG-Glucan-activated NF-κB-like transcription factior to an NF-κB binding site was inhibited by anti-C/EBP-β, but was not prevented by incubation with pre-immune serum, anti-C/EBP-α or anti- C/EBP-y (Figure 8) .
Additional experiments were performed, as described above, to test the ability of the NF-IL6 consensus probe to compete for protein complex formation with our labeled NF- KB consensus probe. The results, shown in Figure 9, show- that for PGG-Glucan-treated cells, cold NF-IL6 probe strongly competed for NF-κB complex formation, while cold mutant NF-IL6 probe did not. These data indicate that, as shown above for BMC2.3 cells, the predominant complex bound to the NF-κB consensus probe in human neutrophils may also contain a member of the NF-IL6 family of proteins.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.