Immune modulatory activity of human ribonucleases

[0001] This application claims the benefit of U.S. Serial No. 60/393,110 filed Jul. 3, 2002, and No. 60/394,511 filed Jul. 10, 2002.

[0002] The invention relates to the field of immunology. In particular it relates to the cytokines stimulated by Rnase family members in leukocytes. Specifically, the invention relates to a novel function of human ribonucleases: immune modulatory activity on leukocytes.

[0003] Ordered arrays of proteins provide an attractive strategy for high-throughput analysis of proteins. To be truly useful for this purpose, however, such arrays must yield sensitive, quantitative, and reproducible measurements of protein levels. It is also desirable that assays on these arrays utilize small sample volumes and be amenable to automated systems for high-throughput processing. There have been a number of recent examples of the use of protein arrays for a variety of applications (1-6). While these approaches have established the feasibility of protein arrays, they have not yet demonstrated practical utility for measuring protein expression levels in a manner analogous to a gene expression array. A microarray consisting of immobilized antibodies is the most straightforward near-term approach for developing a chip for highly parallel analysis of protein levels. Experience with such arrays is limited, and the levels of sensitivity (ca. 10 ng/mL) and multiplexing have been insufficient for quantifying most biological change (7-10).

[0004] Human extra cellular ribonucleases (RNases) are widely distributed in various organs and body fluids and together with other members of the mammalian RNase A superfamily, can be classified into four different RNase families on the basis of their structural, catalytic and/or biological properties 1. According to this classification, human ribonucleases found not only in pancreas but also in other tissues and fluids, and characterized by sequence, structural and catalytic properties similar to those of bovine or human pancreatic RNases, belong to the mammalian pancreatic-type (pt) RNase family. Consequently, the extracellular ribonucleases expressed in tissues other than pancreas and also found in several fluids, and characterized by sequence and catalytic properties similar to those of bovine kidney RNase k2 or human eosinophil-derived neurotoxin (EDN)/liver RNase, constitute the nonpancreatic-type (npt) RNase family. Other members of the RNase A superfamily (for example human plasma RNase 4, bovine liver RNase BL 4 and porcine liver RNase PL 3), being structurally more similar to mammalian ptRNases but sharing some catalytic properties with both pt and npt ribonucleases, have been grouped into a third distinct RNase family and referred to as pt/nptRNases. Human angiogenin (an a typical ribonuclease distinguished by its potent angiogenic action linked to a weak unusual ribonucleolytic activity) may constitute, together with other mammalian angiogenins, a fourth RNase family whose members could be designated as angRNases 1.

[0005] In addition to their RNase activity, several RNases have been shown to have special biological actions, i.e., antitumor, antiviral and angiogenic properties. The mechanism(s) by which this occurs are unknown. Two eosinophil granule proteins, eosinophil-derived neurotoxin (EDN, nptRNase 2) and cosinophil cationic protein (ECP, nptRNase 3), possess RNase activities 2. ECP and EDN exhibit antiviral properties that parallel but are not fully explained by their RNase action 2; 3; 4. ECP stimulates histamine release by rat mast cells 5, ICAM-1 expression by cultured human nasal epithelial cells 6, and increases vascular permeability in the hamster cheek pouch model 7. ECP also stimulates histamine, tryptase and prostaglandin D2 release by human cardiac mast cells 8, a concentration-dependent release of lactoferrin from explants of human bronchi and release of mucins by both feline and human tracheal explants. ECP has been reported to enhance the expression of the receptor for insulin growth factor I on human bronchial epithelial cell line 9. ECP inhibits the constitutive immunoglobulin synthesis by two human lymphoblastoid cell lines and by purified human tonsilar B-cells, as well as proliferation of the two cell lines 9 10. The inhibition extends to all immunoglobulin classes and the inhibition of both immunoglobulin synthesis and proliferation are reversed by the addition of IL-4. A similar effect on immunoglobulin synthesis by a human plasma cell line is also observed, and in this instance the inhibition is reversed by IL-6 11.

[0006] Although no direct stimulatory effect of EDN on inflammatory cells has been described, several studies have found modulation of EDN by numerous cytokines. Eotaxin has been shown to prime normal human eosinophils for exaggerated EDN release stimulated by Substance-P 12. Eotaxin significantly induces EDN release in a dose-dependent manner, indicating that eotaxin may play an important role not only as a selective chemotactic factor for eosinophils but also as a secretagogue 13. Cultured eosinophils degranulate EDN induced by sIgA-beads 14, and EDN release by IL-5-treated cosinophils reaches plateau after 12 h 15. Ex vivo IL-5 production significantly correlates with the number of airway eosinophils and levels of EDN and IL-5 in bronchoalveolar lavage fluid cells treated with budesonide 16. CD34 peripheral blood progenitor cells grown with cytokines promoting eosinophil differentiation produce EDN 17. Eosinophil-inducible human myeloid cell line can be stimulated by a combination of IL-3, GM-CSF and IL-5 to produce all the eosinophil granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), ECP, EDN, and the Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase) 18. Immune complexes (secretory IgA, IgG, IgE) are known as potent triggering stimuli of eosinophil degranulation as well as complement fragments (C3b, C3bi). Cytokines (IL-5, GM-CSF), PAF and peptides (substance P) act both as weak degranulation inducer and degranulation enhancer 19. The release of EDN has been measured by RIA as an index of degranulation 20. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. These results suggest that cytokines which induce eosinophil differentiation and proliferation also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation. Therefore, numerous studies have established a link between Rnases (EDN and ECP) and immunoregulatory molecules.

[0007] Cellular receptors for members of the human Rnase superfamily have been described on human endothelial cells and on vascular smooth muscle cells. 25, 26 Various competitors of the binding reaction have also been described, including protamine, heparin, and polylysine. 26 In addition, inbitors of the human Rnases have been described. These include human placental Rnase inhibitor (PRI) and peptides termed chANG and chGNA. 26, 27

[0008] There is a need in the art for improved diagnostic and therapeutic techniques for diseases that are associated with inflammatory processes.

[0009] According to one embodiment of the invention a method is provided for diagnosing an inflammatory syndrome in a patient. The amount of one or more Rnases in a test sample of a patient is determined. The amount determined is compared to an average amount found in control samples from a population of healthy humans. An increased amount in the test sample relative to the average amount indicates an inflammatory syndrome in the patient.

[0010] According to yet another embodiment of the invention a method is provided for treating a patient with an inflammatory syndrome. One or more specific inhibitory molecules selected from the group consisting of antibodies and antisense RNA are administered to a patient with an inflammatory syndrome. The specific inhibitory molecules specifically bind to and inhibit a human Rnase.

[0011] According to yet another embodiment of the invention a method is provided for preventing an inflammatory syndrome in a patient. One or more specific inhibitory molecules selected from the group consisting of antibodies and antisense RNA are administered to an organ or tissue transplant patient at risk of developing an inflammatory syndrome. The specific inhibitory molecules specifically bind to and inhibit a human Rnase. According to still another embodiment of the invention a method is provided for stimulating an immune response. A human Rnase is administered to a subject in need of an augmented immune response. The subject's immune response is increased. Also provided by the present invention is a composition for vaccinating a human. The composition comprises an immunogenic antigen and a human Rnase. In still another embodiment of the invention, a method is provided to monitor the effects of Rnase therapy or anti-Rnase therapy. The amount of one or more enumerated proteins is determined. The one or more proteins are selected from the group consisting of ENA-78, EOT2, BLC, GDNF, 1309, IFN-, IFN-, IL-10, IL-12P40, IL-12p70, IL-13, IL-16, IL-18, IL-1, IL-1ra, IL-2Sra, IL-3, IL-6, IL-6sR, IL-7, IL-8, IP-10, MCP-1, MCP-2, MCP-3, MCSF, MIG, MDC, MIP-1, MIP-1, MPIF-1, NAP-2, RANTES, sCD23, OSM, TARC, TNF-, TNF-R1, uPAR, and fragments thereof. The determination is repeated on a sample collected at a later time. The amounts measured in the samples from the two times are compared. An increased amount over time denotes an effect of an Rnase and a decreased amount denotes an effect of an anti-Rnase therapy. According to yet another embodiment of the invention a method is provided for treating a patient with an inflammatory syndrome. One or more specific inhibitory molecules which specifically bind to a receptor for a human Rnase are administered to a patient. The molecule blocks the human Rnase from binding to its cellular receptor.

[0012] The invention thus provides the art with diagnostic and therapeutic methods for clinically managing inflammatory syndromes.

[0013] FIG. 1. Cartoon of immunoassays with RCA signal amplification:

[0014] (A). In the adaptation of RCA used for protein signal amplification, the 5 end of an oligonucleotide primer is attached to an antibody. (B) The antibody-DNA conjugate binds to its specific target molecule; in the multiplexed microarray immunoassay, the targets are biotinylated secondary antibodies and the conjugate is an antibiotin antibody. (C) A circular DNA molecule hybridizes to its complementary primer on the conjugate, and in the presence of DNA polymerase, and nucleotides, rolling circle replication occurs. (D) A long single DNA molecule that represents a concatamer of complements of the circle DNA sequence is generated that remains attached to the antibody. (E) This RCA product is detected by hybridization of multiple fluorescent, complementary oligonucleotide probes. RCA product fluorescence is measured with a conventional microarray scanning device. The amount of fluorescence at each spot is directly proportional to the amount of specific protein in the original sample.

[0015] FIG. 2. Induction of inflammatory cytokines by Rnase family members resembles cytokine profile following TNF-a treatment:

[0016] CD34 Cells treated with variously reagents as described in Materials and Methods, experiment 1 for 48 hours. Supernatant were harvested and stored at 70 C. before RCA amplified microarray immunoassay.

[0017] The fold of expression was calculated as follows: $\mathrm{fold}=\frac{\mathrm{Cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{treatment}}{\mathrm{cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{medium}\mathrm{only}\mathrm{control}}$

[0018] FIG. 3. Treatment of CD34 immature dendritic cells with different Rnase family members: time course analysis and dose response:

[0019] For dose response experiments, all samples were treated with various concentrations of Rnases for 48 hours. For time course experiment, all samples were treated with 1000 ng/ml Rnases for various hours as indicated.

[0020] FIG. 4. Treatment of monocytes with different Rnase family members: time course analysis and dose response:

[0021] For dose response experiments, all samples were treated with various concentrations of Rnases for 48 hours. For time course experiment, all samples were treated with 1000 ng/ml Rnases for various hours as indicated.

[0022] FIG. 5. Cytokine/Chemokines induced in monocyte cell line by Rnase family members:

[0023] G4 supernatant was harvested from monocytes treated with medium only. For all Rnases, the treatment was 1000 ng/ml for 48 hours.

[0024] Tables:

[0025] Table 1. Levels of Cytokine Expression in CD34 Immature Dendritic Cells

[0026] Mean value of Cy5 fluorescence intensity is presented. The cells were cultured for 48 hours with 1000 ng/ml Rnase 1, hEDN (Rnase 2) and Rnase 3.

[0027] The fold of expression was calculated as follows: $\mathrm{fold}=\frac{\mathrm{Cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{treatment}}{\mathrm{cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{medium}\mathrm{only}\mathrm{control}\left(\mathrm{G4}\right)}$

[0028] Table 2. Levels Cytokine Expression in Monocytes

[0029] Mean value of Cy5 fluorescence intensity is presented. The cells were cultured for 12 hours with 1000 ng/ml Rnase 1, HEDN (Rnase 2) and Rnase 3.

[0030] The fold of expression was calculated as follows: $\mathrm{fold}=\frac{\mathrm{Cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{treatment}}{\mathrm{cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{medium}\mathrm{only}\mathrm{control}\left(\mathrm{G4}\right)}$

[0031] Table 3. Levels of Cytokine Expression in a Monocyte Cell Line

[0032] Mean value of Cy5 fluorescence intensity is presented. The cells were cultured for 48 hours with 1000 ng/ml Rnase 1, HEDN (Rnase 2) and Rnase 3.

[0033] The fold of expression was calculated as follows: $\mathrm{fold}=\frac{\mathrm{Cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{treatment}}{\mathrm{cy5}\mathrm{fluorescence}\mathrm{value}\mathrm{of}\mathrm{medium}\mathrm{only}\mathrm{control}\left(\mathrm{G4}\right)}$

[0034] It is a discovery of the present inventors that members of the human ribonuclease family induce expression of certain sets of cytokines in human leukocytes. Based on the function of these induced cytokines, it is suggested that human ribonuclease family members have novel immune modulatory activity. The ribonucleases may be any selected from the following families: pancreatic-type (pt) RNase family, nonpancreatic-type (npt) RNase family, pt/nptRNases, and angRNases. Particularly useful are Rnase 1, HEDN (Rnase 2), and Rnase 3 (ECP).

[0035] Inflammatory syndromes that can be advantageously diagnosed and treated according to the present invention include sepsis, arthritis, allergy, enteritis, severe acute pancreatitis, emphysema, multiple organ failure, tissue or organ rejection, cardiovascular disease, infectious disease, autoimmune disease, rheumatoid arthritis, psoriasis, lupus, inflammatory bowel disease, and acute respiratory distress syndrome (ARDS). Other inflammatory syndromes are also amenable to the methods of the invention.

[0036] Test samples used for performing the diagnostic method are preferably from serum, plasma, blood, lymph fluid, peripheral lymphatic tissue, or blood. Desirably the test sample contains, or has contained, leukocytes, monocytes, dendritic cells, or Langerhans cells. However, it may be desirable that the actual sample upon which the assay is performed be relatively free of cells.

[0037] Altered expression of a cytokine can be determined relative to a control sample. The control sample can be obtained from an organ distal to the area of local inflammation in the test subject. Alternatively the control sample can be obtained from a subject or subjects not experiencing or evidencing an inflammatory syndrome. An average value or range can be determined from a population of healthy individuals and used as a control value. Altered expression can be determined at any threshold that is statistically significant. This can be an increase relative to a control sample of 25%, 50%, or 75%, for example. The threshold can be set to at least two-fold the level of the control sample. Alternatively, the threshold can be set to at least three-fold the level in the control sample. A more stringent threshold can be set to at least four-fold the level in the control sample.

[0038] Altered expression of a cytokine can be determined using either mRNA or protein as an indication of expression level. Preferably the protein will be determined. The determination need not be strictly quantitative. For example, in cases where a cytokine goes from an unexpressed to an expressed state a qualitative assessment may be sufficient. Any assay known in the art for detecting gene expression can be used, either individually or multiplexed. The assays used may involve gene arrays, protein arrays, antibody arrays, Western blotting, ELISA, immunoprecipitation, filter binding assays, hybridization assays, etc. The protein microarray employing a rolling circle amplification for detection described in detail below is preferred, but need not be used. Briefly, capture antibodies are affixed to a solid support in a predetermined pattern (array) and test sample is applied to the array so that proteins (cytokines) in the test sample can bind to antibodies on the array which are specific for that particular protein. Second antibodies are applied which are specific for the same set of proteins as are the capture antibodies. The second set of antibodies can be labeled with a hapten. A third set of antibodies is then applied to the array. The third set of antibodies is specific for the hapten on the second set of antibodies or with the constant region of the second set of antibodies. The third set of antibodies contains an attached oligonucleotide. The oligonucleotide can be used as a primer to amplify a template to create an amplification signal. Preferably the template is a circular DNA such that rolling circle amplification can create a large signal. Alternatively, the second antibody can be directly detectable, for example by rolling circle amplification of an attached oligonucleotide.

[0039] Unwanted immune reactions associated with inflammatory syndromes can be treated by administering an antibody which specifically binds to a human Rnase. The antibody can be a monoclonal or polyclonal antibody. It can be a complete antibody molecule or a fragment. Standard antibody fragments are known in the art and any of these can be used, including Fab, F(ab)₂. Single chain Fv (ScFv) can also be used. The antibodies can if desired be attached to other moieties, such as therapeutic agents. Single antibodies or cocktails of antibodies can be used. The cocktails can be directed to the same or different cytokines. Antibodies can be administered by any means known in the art, including but not limited to intravenous, intrathecal, directly to the thymus or to a lymph nodes, subcutaneous, oral, and intramuscular. Antisense molecules can also be used which specifically bind to mRNA encoding an Rnase and inhibit expression of an Rnase.

[0040] Rnasel, HEDN (Rnase 2) and Rnase 3-treated leukocytes (CD34 cell and monocytes) expressed a set of cytokine, chemokines growth factors and soluble receptors, including ENA-78, EOT2, BLC, GDNF, 1309, IFN-, IFN-, IL-10, IL-12P40, IL-12p70, IL-13, IL-16, IL-18, IL-1, IL-1ra, IL-2Sra, IL-3, IL-6, IL-6sR, IL-7, IL-8, IP-10, MCP-1, MCP-2, MCP-3, MCSF, MIG, MDC, MIP-1, MIP-1, MPIF-1, NAP-2, RANTES, sCD23, OSM, TARC, TNF-, TNF-R1 and uPAR. Of cytokines that were induced, IL-6, MIP-1, MIP-1 and TNF- are known to play a critical role in mediation of inflammatory response; ENA-78, MCP-1, MCP-2, MCP-3, MIP-1, MIP-1-, I309, IP-10 and Rantes belong to Chemokine family. hEDN and Rnase 1 resemble TNF- in inducing secretion cytokine expression. These induced cytokines and chemokines are known to play important roles in various aspects of host defense. Many of these cytokine/chemokines have been detected in a wide variety of disease states involving inflammation including, but not limited to angiogenesis, tissue injury, autoimmunity and neoplastic tissue.

[0041] Antibodies and anti-sense molecules can be administered by any technique known in the art. Such methods include, but are not limited to intravenous, intramuscular, subcutaneous, oral, nasal and intrabronchial injections or instillations.

[0042] Compositions for vaccinating individuals can be any standard immunogenic formulation which contains an antigen of choice. Other formulation components can be present including excipients, stabilizers, and adjuvants. The selected one or more human Rnase is present in an effective amount for stimulating an immune response beyond the response level when the Rnase is not present. Determination of the proper dosage is well within the skill of the ordinary artisan.

[0043] Rnases can also be administered to other individuals in need of an immune adjuvant. Such individuals include those who are immunocompromised. Individuals who are immunocompromised include those who have been subjected to a the side effects of drugs or radiation, those who have been subjected to toxic substances present in the environmental or workplace, and those who have diseases which diminish the natural immune responses.

[0044] To date there are no reports that specific cytokines/chemokines can be induced by Rnase family members in vivo or in vitro systems. However, many reports have described certain cytokines can induce Rnases expression. In addition to their ribonuclease activities, Rnases possess special biological properties, such as neurotoxicity, angiogenic activities, immunosuppressivity, anti-tumor and anti-viral activities. However, the fundamental mechanism underlying these important biological activities is unclear. This discovery of a specific sets cytokines/chemokines induced by Rnase 1, hEDN or Rnase 3 is very important to help us understanding these biological activities of Rnase family.

[0045] We utilized highly sensitive antibody based microarray protein chips, which detect 78 cytokines/chemokines simultaneously. Our result demonstrated that a specific set of cytokines/chemokines was induced in Rnase 1, HEDN or Rnase 3 treated immature dendritic cells, monocytes and a monocyte cell line. The profile of cytokines/chemokines induced by Rnase 1, hEDN or Rnase 3 may reveal mechanism of Rnases actions and help us understand the why Rnases have anti-tumor, anti-vial, angiogenic activities.

[0046] hEDN, Rnase I, Rnase 3 or other members of Rnase family are therapeutic targets. Inhibitors (in the form of antibodies, small molecular drugs, anti-sense RNA therapy) of hEDN and Rnase 1 and members of the hEDN/Rnase 1 like family can be used to treat inflammatory diseases in general including, but not limited to infectious diseases, acute/and or chronic inflammation and autoimmune disorder as well as transplantation situations. Specifically such conditions include sepsis, cardiovascular disease, infectious disease, cancer, rheumatoid arthritis, multiple organ failure, acute respiratory distress syndrome (ARDS), psoriasis, lupus, inflammatory bowel disease, and organ or tissue transplant rejection. Anti-hEDN and anti-Rnase 1 can also be used as drugs to treat diseases associated with elevated hEDN and Rnase 1 expression. Similarly, agents which bind to the cellular receptor for these Rnase family members thereby competing or blocking the binding of the Rnase family member can be used as therapeutic agents.

[0047] Therapeutics based on inhibition Rnase family members can take the form of proteins, antibody-based therapy or small molecular drugs, anti-sense RNA therapies. The receptors for Rnase family members can also be considered as therapeutic targets for protein therapy, antibody therapy or small molecular drug therapy. As Rnase family members resemble TNF- in their ability to induce cytokine/chemokine expression in leukocytes, inhibitors of Rnase family members (protein therapeutics, antibody targets or small molecular drugs, anti-sense therapies), can be used in inflammatory diseases situations as well as in transplantation situations where anti-TNF-a has been shown to be effective.

[0048] The levels of 78 cytokines were measured in 16 cell culture supernatants. The treatments are described in Materials and Methods. RNase 1 and hEDN (Rnase 2) induce a specific subset of cytokines/chemokines in dendritic cells including ENA-78; IL-12p40, Il-2sRa, IL-6, MCP-2, MCP-3, MIP1, MIP1- MPIF and Rantes. The profile of cytokines induced by Rnase family members resembled to cytokine profile following TNF- treatment (FIG. 2). However, cytokine profiles following treatment with Rnase family members and TNF- were not completely overlapping. This result suggested the overlapping but distinct functions of Rnase family members and TNF-.

[0049] Cytokines/Chemokines induced in dendritic cells by Rnase family members including ENA-78; IL-12p40, Il-2sR, IL-6, MCP-2, MCP-3, MIP1a, MIP1b MPIF and Rantes was confirmed by the second set of experiments. In addition, the second set of experiments also examined the dependence of this response on RNase concentration, enzymatic activity, treatment time, cell-type specificity and different sources of RNases protein preparation. In addition to previously tested RNase 1 and hEDN, another eosinophil associated RNase, RNase 3 and Rnase 4 were also examined.

[0050] In CD34 cells, 18 cytokines (ENA-78, 1-309, IL-12p40, IL-12p70, IL-6, IL-7, IP-10, MCP-1, MCP-2, MCP-3, MCSF, MIG, MIP1, MPIF-1, NAP-2, Rantes, TNF- and TNFRI) were induced by Rnase 1, 13 cytokines (ENA-78, 1-309, IL-12p40, IL-6, IL-7, IP-10, MCP-1, MCP-2, MCP-3, MIP1, Rantes, sCD23 and TNF) were induced by hEDN, 3 cytokine (IL-6, ENA-78 and MCP-3) were induced by Rnase 3 (table 1). Cytokines with induction folds 3 (comparing to G4 medium treated cells) were counted. The results confirmed that similar set of pro-inflammatory cytokines was induced by three Rnases. Furthermore, the responses were dependent on Rnases treatment time and concentrations (FIG. 3).

[0051] The expression level peaked at different time point for different cytokines. For example, with 1000 ng/ml Rnase 1, the expression of IL-6, MIP1, Rantes and TNF peaked at 6 hours, the expression of ENA-78, IP-10, MCP-1 and 1-309 peaked at 12 hours, the expression of MCP-2, MCP-3, peaked at 24 hours, the expression of IL-12p40 peaked at 48 hours (FIG. 3). The sequential order of cytokine induced implied molecular mechanism of Rnases action. The cytokine induced at earlier stage might stimulate CD 34 cells to produced cytokines in later stages. This data is consistent with our hypothesis that Rnases acted upstream of TNF-, yet the functions of Rnase are not identical to TNF-. Of these cytokine induced at early stage, IL-6 has been described as both a pro-inflammatory and anti-inflammatory molecule, a modulator of bone resorption, a promoter of hematopoiesis, and an inducer of plasma cell development; TNF- plays a critical role in mediation of the inflammatory response and in mediation of resistance to infections and tumor growth; MIP1 and Rantes are CXC chemokines that chemoattract and activate monocytes, dendritic cells, T-lymphocytes, natural killer cells, B-lymphocytes, basophils, and eosinophils.

[0052] Monocytes expressed similar set of pro-inflammatory cytokines upon the treatment with Rnase family members. Table 2 summarized the expression of all cytokines after 12 hours of incubation with 1000 ng/ml Rnases. 16 cytokines (EOT2, 1-309, IFN-, IL-10, IL-12p40, IL-13, IL-6, IL-7, IP-10, MCP-2, MIG, MIP1, MIP-1, MPIF-1, Rantes and TNF-) were induced by Rnase 1; 7 cytokines (EOT2, IL-16, IL-6, MIP1, MPIF-1, Rantes and IP-10) were induced by hEDN (Rnase 2), 2 cytokines (MCP-1 and MIP-1) were induced by Rnase 3. Again, cytokines with induction folds >3 (comparing to G4 medium treated cells) counted.

[0053] We also observed the similar responses to treatment time and concentration (FIG. 4). Similarly, the level of expression for each cytokine peaked at different time points (FIG. 4). Upon culture with 1000 ng/ml Rnase 1, Il-6, MIP-10, MIP-1, MCP-1, MCP-2, Rantes and TNF- expression peaked after 6 hours incubation; Rantes peaked at 12 hours; 1309 and IP-10 peaked at 48 hours. This result suggested that Rnase family members could induce pro-inflammatory cytokines in general across a broad range of cell types, with each cell type having slightly different specific responses.

[0054] Under the condition of 1000 ng/ml and 48 hours treatment, Rnase 1, HEDN (Rnase 2) and Rnase 3 stimulated similar yet distinct sets of cytokines (see table 3). 28cytokines (BLC, 1309, IFN-, IFN-, IL-10, IL-12P40, IL-13, IL-18, IL-10, IL-1ra, IL-2Sra, IL-3, IL-6, IL-6sR, IL-8, IP-10, MCP-1, MCP-2, MCP-3, MDC, MIP-1, MIP-1, NAP-2, OSM, TARC, TNF-, TNF-R1 and uPAR) were induced by Rnase 1; Il cytokines (GDNF, IFN-, IL-10, IL-18, IL-1, IL-6, IL-8, IP-10, MCP-2, MDC and MIP-1) were induced by hEDN (Rnase 2) and 4 cytokines were induced by Rnase 3 (GDNF, IFN-, IL-10, and IL-13 (FIG. 5). Since this cell line has been cultured in vitro for long time, it has unique responses.

[0055] In Experiment 1, 16 cell culture supernatants (RPMI supplemented with GMCSF and IL-4) were provided by Drs. De Yung and Zack Howard of NCI (NCI Frederick, Frederick, Md. 21702). The cells had been treated as follows:

[0056] 1. Medium alone without cells, a background control.

[0057] 2. Medium with cells, a negative control.

[0058] 3. LARC at 100 ng/ml, human chemokine.

[0059] 4. hBD2 at 1000 ng/ml, human beta defensin 2.

[0060] 5. hBD3 at 1000 ng/ml, human beta defensin 3.

[0061] 6. PARC at 1000 ng/ml, human chemokine.

[0062] 7. hNPm at 1000 ng/ml, natural human neotrophil defensins (a), mixture of hNP1, hNP2 and hNP3 and isolated from the granules of polymorphonuclear leukocyte.

[0063] 8. hNP1 at 1000 ng/ml, human neutrophil protein, alpha defensin.

[0064] 9. hEDN at 1000 ng/ml, human eosinophil derived neurotoxin.

[0065] 10. mEAR2 at 1000 ng/ml, mouse protein, no effect on human cells and is a negative control.

[0066] 11. RNase1 at 1000 ng/ml, human RNase 1, eosinophil derived, It can strongly activate iDC and is a control for iDC maturation.

[0067] 12. C5a at 10 nM, complement factor 5a.

[0068] 13. W pep. at 100 nM, hexapeptide.

[0069] 14. PAF at 10 ng/ml, platelet activating factor.

[0070] 15. RANTES at 100 ng/ml, human chemokine.

[0071] 16. TNFa at 50 ng/ml, a positive control.

[0072] In Experiment 2, 84 cell culture supernatants (RPMI supplemented with GMCSF and IL-4) were provided by Dr. De Yung and Dr. Zack Howard of NCI (NCI Frederick, Frederick, Md. 21702). Samples were divided into following 5 groups:

[0073] 1. Group 1: (Time-course, 36 samples) monocyte-derived DCs and CD34-derived DCs treated with RNase 1, hEDN (Rnase 2) or RNase 3 for the following times: 0, 2 or 3, 6, 12, 24, and 48 hours.

[0074] 2. Group 2: (Concentration-dependence, 29 samples) monocyte-derived DCs and CD34-derived DCs treated for 48 hrs with 10, 100, 500, or 1000 ng/ml of RNase 1 or hEDN (Rnase 2); or with 1000 or 3000 ng/ml of RNase3.

[0075] 3. Group 3: (RNase activity-dependence, 6 samples) CD34-derived DCs treated with 1000 ng/ml RNase 1 or 2 in the presence of ribonuclease inhibitor.

[0076] 4. Group 4: (Cell type specificity, 8 samples) lymphocytes treated with RNase 1 or hEDN (Rnase 2). Monocyte cell lines treated with RNase 1, hEDN (Rnase 2) or RNase 3.

[0077] 5. Group 5: (independent RNase source, 5 samples) Monocytes treated with 1000 ng/ml RNase 1, hEDN (Rnase 2) or RNase 3.

[0078] Microarray manufacture: Antibody microarrays were printed using a Packard Biosciences (Downers Grove, Ill.) BCA-II piezoelectric microarray dispenser on cyanosilane-coated glass slides divided by Teflon boundaries into sixteen 0.5 cm diameter circular subarrays. Monoclonal antibodies for 78 cytokines (see Supplementary Material for listing of antibodies and vendors) were dispensed in quadruplicate at a concentration of 0.5 mg/ml. Printed slides were blocked as

[0079] and stored at 4 C. until use. Batches of slides were subjected to a quality control consisting of incubation with a fluorescently-labeled anti-mouse antibody, followed by washing, scanning and quantitation. Typically, the coefficient of variability (CV) of antibody deposition in printing was <5%.

[0080] RCA Immunoassay: The assay was performed by a liquid-handling robot (Biomek 2000, Beckman Instruments, Fullerton, Calif., which was enclosed in an 80% humidified, HEPA-filtered, plexiglass chamber. For each sample, duplicates were tested either neat or diluted 1:10. 20 l of samples was applied to each sub-array and immunoassays with RCA signal amplification were performed as described 21 Slides were scanned (GenePix, Axon Instruments Inc., Foster City, Calif.) at 10-m resolution with laser setting of 100 and PMT setting of 550. Mean pixel fluorescence were quantified using the fixed circle method in GenePix Pro 3.0 (Axon Instruments, Foster City, Calif.). The fluorescence intensity of 8 microarray features (duplicates subarrays and quadruplicates spots in each subarray) was averaged for each feature and sample, and the resulting cytokine values were determined. For every slide, a set of blanks was run as a negative control.

[0081] Data Quality Control: Subarray(s) were excluded from analysis if fluorescent intensities were generally weak (indicating weak RCA in that particular subarray), if there were visible defects in the array (such as scratches), or if there was high background signal. A total of 168 subarrays (84 samples) were analyzed, 4 subarray-1s and 4 subarray-2s were excluded on the basis of these quality control criteria. None of the samples failed both duplicated subarrays. Analyses were performed using complete set of data containing the levels of all 78 cytokines from 84 cell culture supernatants. Untransformed fluorescent intensities were used as data values in all of the analyses.

TABLE 1
Levels of cytokine expression in CD34 cell
(with 1000 ng/ml Rnases for 48 hours)
			Rnase 1	Rnase 2	Rnase 3	fold	fold	fold (Rnase
	Cytokine	G4 Mean	Mean	Mean	Mean	(Rnase 1/G4)	(hEDN/G4)	3/G4)
1	ANG	31415	36129	33853	35432	1	1	1
2	AR	302	364	443	227	1	1	1
3	BDNF	144	201	181	94	1	1	1
4	BLC	63	48	36	59	1	1	1
5	CNTF	151	141	186	164	1	1	1
6	EGF	89	122	104	118	1	1	1
7	ENA-78	150	13673	3824	391	91	26	3
8	Eot	383	65	57	89	0	0	0
9	Eot2	61666	61526	62290	61996	1	1	1
10	Fas (PM)	1721	1984	1916	1249	1	1	1
11	FGF-6	106	148	147	89	1	1	1
12	FGF-7	60	28	15	64	0	0	1
13	FGF-9	181	77	53	189	0	0	1
14	Flt3Lig	99	161	137	63	2	1	1
15	G-CSF (PM)	111	148	161	70	1	1	1
16	GDNF	182	130	159	182	1	1	1
17	GM-CSF	61146	62553	62748	62071	1	1	1
18	HCC4	96	120	117	82	1	1	1
19	I-309	8875	31597	30261	9828	4	3	1
20	IFN-a (EDG)	64	36	39	94	1	1	1
21	IFN-g	723	807	815	897	1	1	1
22	IL-10 (EDG)	24	20	52	11	1	2	0
23	IL-11	1138	1178	1264	931	1	1	1
24	IL-12 p40	147	4718	654	142	32	4	1
25	IL-12 p70 (PM)	33	221	61	38	7	2	1
26	IL-13 (PM)	15	21	27	16	1	2	1
27	IL-15	183	164	70	198	1	0	1
28	IL-16	98	118	139	83	1	1	1
29	IL-17	1798	2173	2715	1698	1	2	1
30	IL-18	64	37	31	44	1	0	1
31	IL-1a	2203	1983	2184	1397	1	1	1
32	IL-1b	266	535	340	200	2	1	1
33	IL-1ra	10077	19903	22104	9895	2	2	1
34	IL-1sR1	116	195	134	87	2	1	1
35	IL-2	208	268	254	184	1	1	1
36	IL-2sRa (PM)	154	202	151	186	1	1	1
37	IL-3 (PM)	86	29	47	42	0	1	0
38	IL-4	63960	64361	64653	64446	1	1	1
39	IL-5	108	81	137	79	1	1	1
40	IL-6	319	58054	23689	1049	182	74	3
41	IL-6sR	1596	2131	2383	941	1	1	1
42	IL-7 (PM)	154	1656	552	232	11	4	2
43	IL-8	60009	64332	63952	62615	1	1	1
44	IP-10 (PM)	322	23690	3319	352	74	10	1
45	LIF	235	271	298	227	1	1	1
46	MCP-1	15153	58286	56164	17666	4	4	1
47	MCP-2 (PM)	2176	55975	18279	4098	26	8	2
48	MCP-3 (PM)	274	26568	5030	704	97	18	3
49	M-CSF	222	1088	482	191	5	2	1
50	MDC	45237	36810	38647	26001	1	1	1
51	MIF	146	167	156	188	1	1	1
52	MIG (PM)	115	448	221	96	4	2	1
53	MIP-1a	525	7341	3757	1070	14	7	2
54	MIP-1b	30508	59783	56941	27850	2	2	1
55	MIP-1d	404	552	672	243	1	2	1
56	MPIF-1	301	953	530	559	3	2	2
57	MSP	129	132	124	173	1	1	1
58	NAP-2	75	269	130	121	4	2	2
59	NT3	155	186	191	142	1	1	1
60	NT4	178	218	174	158	1	1	1
61	OSM	9904	18701	17521	13797	2	2	1
62	PARC	21095	19865	21571	19728	1	1	1
63	P1GF	153	71	105	110	0	1	1
64	Rantes	1936	41979	22857	1477	22	12	1
65	sCD23 (PM)	1633	4073	4625	1841	2	3	1
66	SCF	111	137	139	101	1	1	1
67	SDF-1a	430	655	746	294	2	2	1
68	sgp130	123	263	218	106	2	2	1
69	TARC	13914	14432	18429	14262	1	1	1
70	TGF-b1	307	394	454	347	1	1	1
71	TGF-b3	81	54	72	67	1	1	1
72	TNF-a	189	4744	855	168	25	5	1
73	TNF-b	261	355	351	256	1	1	1
74	TNF-R1	254	829	398	150	3	2	1
75	TNF-RII (Bio)	388	434	365	141	1	1	0
76	TRAIL (PM)	29	23	43	16	1	1	1
77	uPAR	11204	23963	22325	11292	2	2	1
78	VEGF	378	446	339	73	1	1	0

[0082]

TABLE 2
Levels of cytokine expression in monocytes
(with 1000 ng/ml Rnases for 12 hours)
		0 Hours	12 hours	0 Hours	12 hours	0 Hours	12 hours	Fold at 12	Fold at 12	Fold at 12
		Mean Rnase	Mean	Mean	Mean	Mean	Mean	hours	hours	hours
	Cytokine	1	Rnase 1	Rnase 2	Rnase 2	Rnase 3	Rnase 3	Rnase 1	Rnase 2	Rnase 3
1	ANG	58586	56597	62021	62680	45856	44777	1	1	1
2	AR	1284	866	717	885	443	457	1	1	1
3	BDNF	389	170	279	255	154	190	0	1	1
4	BLC	144	150	113	102	83	103	1	1	1
5	CNTF	203	291	243	232	221	302	1	1	1
6	EGF	188	270	162	156	184	209	1	1	1
7	ENA-78	129	216	133	124	110	181	2	1	2
8	Eot	96	116	107	82	89	127	1	1	1
9	Eot2	2809	8501	2679	7589	1811	1915	3	3	1
10	Fas (PM)	4359	5157	4523	4271	3093	5163	1	1	2
11	FGF-6	618	175	275	301	164	132	0	1	1
12	FGF-7	129	114	104	60	71	81	1	1	1
13	FGF-9	358	362	341	226	253	311	1	1	1
14	Flt3Lig	265	88	179	147	123	92	0	1	1
15	G-CSF (PM)	385	248	256	174	141	107	1	1	1
16	GDNF	118	288	237	210	219	361	2	1	2
17	GM-CSF	64507	63294	64329	64736	64236	63714	1	1	1
18	HCC4	197	126	153	108	111	115	1	1	1
19	I-309	8458	36065	7695	10860	7496	9206	4	1	1
20	IFN-a (EDG)	34	182	1709	82	108	98	5	0	1
21	IFN-g	530	907	814	626	771	1156	2	1	2
22	IL-10 (EDG)	14	215	42	19	25	35	16	0	1
23	IL-11	2417	1578	1332	1946	959	2104	1	1	2
24	IL-12 p40	269	893	267	237	188	165	3	1	1
25	IL-12 p70 (PM)	104	202	121	81	50	58	2	1	1
26	IL-13 (PM)	31	91	106	75	41	53	3	1	1
27	IL-15	231	384	281	228	224	322	2	1	1
28	IL-16	760	251	244	798	94	183	0	3	2
29	IL-17	4662	3800	3208	4114	2491	4466	1	1	2
30	IL-18	249	271	104	175	129	164	1	2	1
31	IL-1a	6191	5614	5590	5961	3903	4529	1	1	1
32	IL-1b	585	388	377	242	323	352	1	1	1
33	IL-1ra	13775	10371	6995	9584	2791	4088	1	1	1
34	IL-1sR1	300	198	164	130	135	127	1	1	1
35	IL-2	407	189	245	228	207	267	0	1	1
36	IL-2sRa (PM)	191	377	220	214	210	286	2	1	1
37	IL-3 (PM)	40	98	189	58	81	79	2	0	1
38	IL-4	65168	64663	65292	65224	64911	65265	1	1	1
39	IL-5	111	247	133	143	161	226	2	1	1
40	IL-6	155	28087	139	2023	150	157	181	15	1
41	IL-6sR	5972	2544	3265	4682	844	1063	0	1	1
42	IL-7 (PM)	498	1937	666	1447	844	1136	4	2	1
43	IL-8	65369	64334	64746	65199	63918	65226	1	1	1
44	IP-10 (PM)	674	45171	382	2303	299	447	67	6	1
45	LIF	819	362	454	437	330	491	0	1	1
46	MCP-1	1394	2592	791	1172	463	1247	2	1	3
47	MCP-2 (PM)	222	4078	180	237	190	195	18	1	1
48	MCP-3 (PM)	134	171	97	99	124	170	1	1	1
49	M-CSF	523	271	349	290	214	324	1	1	2
50	MDC	62184	59209	56313	56438	46926	51340	1	1	1
51	MIF	2677	2835	1608	1733	511	531	1	1	1
52	MIG (PM)	348	2139	247	369	106	161	6	1	2
53	MIP-1a	246	2353	274	633	356	431	10	2	1
54	MIP-1b	1054	64123	1411	26215	647	2024	61	19	3
55	MIP-1d	1284	1864	1327	1225	626	783	1	1	1
56	MPIF-1	147	850	233	1359	171	182	6	6	1
57	MSP	140	340	198	271	254	371	2	1	1
58	NAP-2	205	341	195	194	146	141	2	1	1
59	NT3	460	84	308	308	144	249	0	1	2
60	NT4	438	25	268	231	121	230	0	1	2
61	OSM	3412	7474	2429	4791	3077	5203	2	2	2
62	PARC	39668	37686	26231	41979	19550	19756	1	2	1
63	P1GF	373	366	371	349	343	215	1	1	1
64	Rantes	982	42944	568	3846	600	878	44	7	1
65	sCD23 (PM)	12194	15058	7959	10574	4751	2597	1	1	1
66	SCF	292	13	243	156	136	182	0	1	1
67	SDF-1a	1133	914	885	812	470	669	1	1	1
68	spg130	591	374	380	341	151	159	1	1	1
69	TARC	37478	31119	29304	31940	26218	34407	1	1	1
70	TGF-b1	618	273	346	469	320	502	0	1	2
71	TGF-b3	82	130	90	87	109	111	2	1	1
72	TNF-a	358	3605	228	330	150	217	10	1	1
73	TNF-b	422	312	333	354	171	396	1	1	2
74	TNF-R1	1066	640	423	779	322	255	1	2	1
75	TNF-RII (Bio)	1616	848	1040	1118	927	867	1	1	1
76	TRAIL (PM)	27	57	71	24	53	62	2	0	1
77	uPAR	23330	22982	15587	26346	12471	13294	1	2	1
78	VEGF	1032	705	850	999	695	801	1	1	1

[0083]

TABLE 3
Monocyte cell line responses
(with 1000 ng/ml Rnases for 48 hours)
						Rnase	Rnase	Rnase
		G4	Rnase 1	Rnase 2	Rnase 3	1/G4	2/G4	3/G4
	Cytokine	Mean	Mean	Mean	Mean	Folds	Folds	Folds
1	ANG	3245	4627	2607	2564	1	1	1
2	AR	783	896	623	403	1	1	1
3	BDNF	438	500	295	498	1	1	1
4	BLC	175	6859	222	249	39	1	1
5	CNTF	708	670	434	483	1	1	1
6	EGF	304	390	244	242	1	1	1
7	ENA-78	241	384	169	210	2	1	1
8	Eot	264	299	140	225	1	1	1
9	Eot2	29468	61933	40578	15353	2	1	1
10	Fas (PM)	5067	4964	4072	3888	1	1	1
11	FGF-6	217	353	205	175	2	1	1
12	FGF-7	159	232	132	92	1	1	1
13	FGF-9	643	618	419	596	1	1	1
14	Flt3Lig	153	254	121	131	2	1	1
15	G-CSF (PM)	352	617	288	359	2	1	1
16	GDNF	138	321	361	353	2	3	3
17	GM-CSF	41514	52466	51602	49628	1	1	1
18	HCC4	215	220	169	157	1	1	1
19	I-309	139	25497	233	96	183	2	1
20	IFN-a (EDG)	16	159	141	136	10	9	9
21	IFN-g	426	1241	858	1008	3	2	2
22	IL-10 (EDG)	35	199	103	156	6	3	5
23	IL-11	2397	2579	2499	2076	1	1	1
24	IL-12 p40	179	4467	293	219	25	2	1
25	IL-12 p70 (PM)	85	125	56	86	1	1	1
26	IL-13 (PM)	18	81	37	57	5	2	3
27	IL-15	271	468	366	450	2	1	2
28	IL-16	208	289	174	165	1	1	1
29	IL-17	4865	5434	4018	3682	1	1	1
30	IL-18	139	1312	428	345	9	3	2
31	IL-1a	5897	8681	4311	4441	1	1	1
32	IL-1b	594	36358	2492	524	61	4	1
33	IL-1ra	2238	17602	2135	1883	8	1	1
34	IL-1sR1	213	253	195	157	1	1	1
35	IL-2	376	419	224	439	1	1	1
36	IL-2sRa (PM)	152	455	354	276	3	2	2
37	IL-3 (PM)	60	210	96	94	3	2	2
38	IL-4	63496	59183	60579	47841	1	1	1
39	IL-5	227	326	230	145	1	1	1
40	IL-6	138	7618	442	194	55	3	1
41	IL-6sR	3284	9718	5938	3980	3	2	1
42	IL-7 (PM)	1524	749	436	455	0	0	0
43	IL-8	4223	58445	33807	2989	14	8	1
44	IP-10 (PM)	464	18336	2862	466	40	6	1
45	LIF	634	656	640	667	1	1	1
46	MCP-1	16012	62453	31760	10120	4	2	1
47	MCP-2 (PM)	414	61580	3094	376	149	7	1
48	MCP-3 (PM)	195	40099	232	279	206	1	1
49	M-CSF	330	388	332	365	1	1	1
50	MDC	502	50360	1667	407	100	3	1
51	MIF	928	1951	1078	665	2	1	1
52	MIG (PM)	166	306	192	147	2	1	1
53	MIP-1a	661	50643	913	885	77	1	1
54	MIP-1b	4479	62477	33707	4270	14	8	1
55	MIP-1d	832	1090	729	788	1	1	1
56	MPIF-1	17187	11862	9430	18049	1	1	1
57	MSP	416	371	293	478	1	1	1
58	NAP-2	81	1003	129	159	12	2	2
59	NT3	188	244	276	196	1	1	1
60	NT4	330	375	321	220	1	1	1
61	OSM	736	10650	690	657	14	1	1
62	PARC	339	510	204	205	2	1	1
63	P1GF	777	700	620	984	1	1	1
64	Rantes	63743	63160	63898	61081	1	1	1
65	sCD23 (PM)	4502	10886	6206	2593	2	1	1
66	SCF	207	270	161	168	1	1	1
67	SDF-1a	1596	1719	1005	1143	1	1	1
68	sgp130	183	368	238	107	2	1	1
69	TARC	208	8173	258	200	39	1	1
70	TGF-b1	631	798	477	571	1	1	1
71	TGF-b3	411	354	274	353	1	1	1
72	TNF-a	306	33486	672	242	109	2	1
73	TNF-b	341	507	381	383	1	1	1
74	TNF-R1	2045	9200	2010	974	4	1	0
75	TNF-RII (Bio)	1006	1084	875	568	1	1	1
76	TRAIL (PM)	89	100	81	91	1	1	1
77	uPAR	1842	23397	3611	1352	13	2	1
78	VEGF	18704	30364	21742	19076	2	1	1

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