NEW METHOD FOR TREATING IMMUNODEFICIENCY OR NEUTROPENIA

Abstract: Neutrophil chemotactic property in melanoma growth stimulatory factor and its utility for stimulating increased neutrophil number and activity to combat diseases caused by the condition of immunodeficiency resulting from or associated with neutrophil dysfunction or neutropenia are described.

Description: NEW METHOD FOR TREATING IMMUNODEFICIENCY OR NEUTROPENIA The present invention is related generally to the method of treating immunodeficiency. More particularly, the present invention is related to inducing or augmenting neutrophil activity by melanoma growth stimulatory factor (MGSF) . BACKGROUND OF THE INVENTION Melanoma growth stimulatory factor (MGSF) is a growth related protein from B-thromboglobulin (B-TG) superfamily. Although MGSF has a number of properties similar to interleu in-8 (IL-8) , another member of the B- TG family, MGSF has not heretofore been demonstrated to possess neutrophil chemotactic activity. SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to demonstrate neutrophil chemotactic activity of MGSF. It is another object of the present invention to provide a new method for stimulating increased neutrophil number and activity to combat diseases caused by the condition of immunodeficiency resulting from or associated with neutrophil dysfunction or neutropenia. Other objects and advantages will become evident from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings, wherein: Figure 1 shows purification of MGSF receptor/binding protein by ligand affinity binding. 125 I-membrane proteins from Hs294T melanoma cells were allowed to bind to rMGSA- sepharose and eluted as described in Methods. Eluant was concentrated by lyophilization and subjected to reducing SDS-PAGE. Gels were dried and exposed to XAR-5 film for 7 days. Lane a) 125 I-Hs294T membrane preparation eluted from 4- MGSA-sepharose. Lane b) 15 I-Hs294T membrane preparation eluted from sepharose alone. Lane c) 125 I-Hs294T membrane preparation. Figure 2a shows MGSF Binding Curve. Human Hs294T melanoma cells were grown to confluency and incubated in SF culture medium for 48 hours. Cultures were then incubated with 125 I-MGSA and varying concentrations of unlabeled MGSA for 2 hours at 25 β C. Cells were washed 5X on ice, lysed and bound radioactivity was determined by gamma counting. Figure 2b (Inset of Figure 2a) shows Scatchard Analysis of Binding of rMGSF to Hs294T Melanoma Cells. Binding assays of suspension cultures of Hs294T cells were performed with 125 I-rMGSA and unlabeled rMGSF as described in Materials and Methods. Cells were washed once, pelleted through a sucrose cushion and bound radioactivity was counted on a gamma counter. A Scatchard plot of the binding data is shown. Figure 3 shows MGSF/IL-8 Competitive Bindings Curve. Human neutrophils (2xl0 6 ) were suspended in binding medium and incubated with 125 I-IL-8 with or without varying concentrations of unlabeled MGSF or IL-8 for 2 hours at 4°C. Cells were washed once, pelleted through a sucrose cushion, and bound radioactivity was counted on a gamma counter. DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by the demonstration of neutrophil chemotactic activity of the MGSF and making use of this new property of MGSF for inducing or augmenting neutrophil number and activity resulting in increased immune function. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. It is pointed out that a recombinantly made MGSF (rMGSF) and natural MGSF exhibited equivalent growth stimulatory activity. Hence, in the studies described herein, either rMGSF or natural MGSF was employed as found convenient or as noted herein. The specifics of the methods are now described. MATERIALS AND METHODS Preparation of Plasma Membrane-enriched Fraction from Hs294T Melanoma Cells Confluent P-150 dishes (5 per preparation) of Hs294T cells were maintained on serum-free medium for a minimum of 48 hours. After washing with serum-free medium, the cells were removed by scraping and sedimented by centrifugation. The weights of the cell pellets were determined and the cells were resuspended (1:10 w/v) in a hypotonic buffer solution (50 mM mannitol, 5mM Hepes, lOug/ l leupeptin, 1% aprotinin pH 7.4). The cell suspension was passed through an 18 gauge needle, 1 M CaCl 2 was added to this suspension to a final concentration of 10 mM and the suspension was maintained on ice for 10 minutes. The suspension was then passed through a 25 gauge needle five times. Insoluble material was removed by microcentrifugation at 12,500 xg in Eppendorf microfuge tubes for 1 minute at 4°C. The supernatant was ultracentrifuged at 105,000 xg for 90 minutes at 4 β C producing a fine translucent pellet that was resuspended in hypotonic buffer and stored at -80 " C . Iodination of Hs294T Membranes A suspension of Hs294T membranes (10.8 ug) was microfuged for 15 min. at 4 β C. The pellet was resuspended in 2.5 μl 0.5 M sodium phosphate pH 7.2. To the suspended membranes, 20 μl of lactoperoxidase, 10 μl of glucose oxidase, and lOμl 125 I-Na (1 mCi) were added. The reaction was initiated by the addition of 20 μl B-D- glucose (18 mg/ml H 2 0 freshly prepared) followed by incubation of the reagents for 15 min. at room temperature (about 22"-24'C). The membranes were then solubilized by the addition of 1 ml 0.05 M sodium phosphate, .1% Triton X-100 pH 1 :2. This solution was transferred to a PD-10 column that was previously washed with 50 ml H 2 0, 2 ml 0.05 M sodium phosphate, 0.1% Triton X-100, 20 mg/ml bovine serum albumin pH 7.2 followed by 30 ml 0.05 M sodium phosphate, 0.1% Triton X-100 pH 7.2. Material was eluted using 0.05 M sodium phosphate, 0.1% Triton X-100 pH 7.2. Fractions (2 ml) were collected into tubes containing 1 ml 0.05 M sodium phosphate, 0.1% Triton X-100, 20 mg/ml bovine serum albumin pH 7.2. A 5 μl aliquot of each fraction was counted in a gamma counter, then diluted with 2X laemmli electrophoresis sample buffer for visualization by standard SDS-PAGE and autoradiographic analyses. Purification of MGSF Natural MGSF was purified from Hs294T conditioned medium as described by Thomas et al (A. Mol Cell Endo. 57:69-76, 1988.) For some studies recombinant MGSF was used (rMGSF) . The rMGSF was purified from conditioned media from Chinese hamster ovarian (CHO) cells stably transfected with the MGSF gene placed under, the control of the cytomegalovirus promoter as described by Balentien et al, supra. Preparation of Sepharose-bound Recombinant MGSF Cyanogen bromide activated sepharose (0.1 g) was incubated overnight (about 12-16 hrs) with 15 ml ImM HC1. The swollen resin was washed three times with 2.5ml aliquots of 1 mM HC1 then suspended in 375 μl of coupling buffer (98.4 g sodium bicarbonate, 29.2 g sodium chloride per liter water, pH 8.3) . Recombinant MGSF (5 μg per 375 μl coupling buffer) was added to the suspended resin and agitated overnight at 4*C. The resin was pelleted by centrifugation at 500 rpm in a Beck an TJ-6 centrifuge. The supernatant was removed and the resin was washed two times with coupling buffer. The resin was incubated overnight at 4 β C with 1 ml of monoethanolamin . After removing monoethanolamine, the resin was washed with acetate buffer (8.2 g" sodium acetate, 58.4 g sodium chloride per liter water, pH 4.0) followed by Tris buffer (6.075 g Tris, 11.6 g sodium chloride per liter water, pH 8.0). The acetate buffer - Tris buffer cycle was repeated three times. The resin was stored in 0.05 M sodium phosphate buffer, pH 7.2 Binding of 125 I-labelled Hs294T Membranes to rMGSF- Sepharose rMGSF-sepharose was suspended in 30 μl of binding buffer (0.8g sodium chloride, 0.115 g dibasic sodium phosphate, 0.02 g potassium chloride, 0.02 g monobasic potassium phosphate, 0.01 g magnesium chloride, 0.01 g calcium chloride, 1.0 g bovine serum albumin, 1.0 mg leupeptin, and 10,000 kallikrein inhibitory units of aprotinin per 100 ml water, pH 7.2). 125 I-labelled Hs294T membranes (35 μl, -5,000,000 cpm) were added to rMGSA- sepharose and a control sepharose resin. These were incubated at 4'C with agitation for 4 hrs. The resin was pelleted by centrifugation and the supernatant was aspirated. The resins were washed with three X 1.5 ml aliquots of ice cold binding buffer. Material bound to the resins was eluted by incubating with 0.4 ml aliquots 100 mM HC1 for 15 min. After pelleting the resin the supernatant was removed, the elution was repeated and the aliquots were combined and frozen. The solution was dried by speed vac and the material was redissolved in 20 μl 4 mM HC1. Laemmli 2X electrophoresis sample buffer (40 μl) was added, the solutions were boiled for 5 min. , microfuged for 2 min., and the supernatants were electrophoresed in a 7.5% polyacrylamidg gel. After staining with Coomassie blue R-250, the gel was dried and exposed to Kodak XAR5 film for visualization of the radiolabelled proteins. Competitive Bindings Assays MGSF was iodinated by either Bolton-Hunter or the chloramine-T procedure (Bolton et al, Biochem J. 133:529- 539, 1973; Hunter et al. Nature (London) 194, 495 1962). Studies of 125 I-MGSF (specific activity 6.7X10 5 cpm/ng) binding were performed on confluent cultures of Hs294T malignant melanoma cells in 24 well Falcon plates at 25"C. Incubations were performed in a binding buffer containing phosphate buffered saline + 30 mM HEPES + 1% BSA, pH 7.2. After the appropriate length of time, the binding buffer was aspirated from the monolayers and the cells were washed 5X with ice cold binding buffer. The monolayers were then treated with 1 ml of lysis buffer (o.5 g SDS, 0.121 g Tris, 0.037 g EDTA/100 ml H 2 0) at room temperature for 15 min. The lysis buffer was transferred to 12 x 7.5 mm tubes. The wells were rinsed with a second aliquot (0.5 ml) of lysis buffer. The aliquots were combined and the amount of 125 I-MGSF found was determined in a gamma counter. Non-specific binding was determined in the presence of 100 fold excess crude MGSF. Studies of the binding of 125 I-MGSF to several normal and malignant cell lines were performed on suspension cultures (1X10 6 cells/ml) to enable a constant cell number for comparison among cell types. The methods were essentially the same except that cells were recovered by centrifugation between incubations and washes. The lysis step was eliminated. Data are expressed as cpm bound to each cell line divided by cpm bound to Hs294T cells x 100%. MGSF Bioresponse Assay The bioresponse elicited by exogenous MGSF in various cells lines was determined by 3 H-thymidine incorporation assay or in a six-day cell number assay. For tritiated thymidine incorporation assays 10* cells were plated into 96 well paltes (Falcon) in DMEM medium (Gibco) containing 1% fetal bovine serum and incubated overnight at 37°C in a water jacketed C0 2 incubator. The following morning aliquots of MGSF or fetal bovine serum were added, the incubation was continued for an additional 18 hours, and then 5 μCi of 3 H-thymidine was added to each well. Cultures were incubated for an additional 24 hours at which time each of the monolayers was washed twice with 100 μl phosphate buffered saline and fixed with 5% trichloroacetic acid (TCA) for 30 min on ice. The TCA was aspirated and 200 μl of 100% methanol was added to each well. After 30 min the methanol was aspirated and cells were solubilized in 100 μl of 0.3N NaOH for 1 hr ' at 37 β C. Aliquots were added to Beckman Ready Fluor and counted by liquid scintillation counting. For cell number assays, the cells were seeded (5000 cells/well) into 24 well Falcon plates in media containing 10% serum. After a 72-hour incubation, the cells were washed once and placed on serum-free medium. Twenty-four hours later, the medium was aspirated and replaced with serum-free medium with and without aliquots of MGSF. A background count was done on well containing only serum-free medium. On the third and sixth days after growth factor additions, cell number was determined. Those cultures that were not counted on day three were fed at that time. Chemotaxis Assay The neutrophil chemotaxis assay was performed as described by Larsen et al., Science 243:1464-1466, 1989 and Falk et al, J. Immunol. Method. 33:239-247, 1980, except that rMGSF was used. 125 I-IL-8 Competitive Binding Assay Recombinant IL-8 has been expressed in E. coli and purified to homogeneity with specific activity of 2X10 6 ϋ/mg (Furuta et al, J. Biochem. 106:436-441, 1989). Recombinant IL-8 was labelled with 125 I by Bolton-Hunter reagent as previously reported (Samanta et al, J. Exp. Med. 169:1185-1189, 1989; Bolton et al., supra; Falk et al, supra) . Ability of rMGSF to compete with 125 I-IL-8 (1.0 x 10 7 cpm/μg) to human neutrophils was examined. In standard binding assays, 2xl0 6 cells were incubated in duplicate with 4 ng 125 I-IL-8 (40,000 cpm) in RPMI 1640 medium containing 10 mg/ml BSA and 20 mM HEPES buffer pH 7.2 in a total volume of 200 μl. Samples were mixed and incubated at 4 β C for 2 hours. After incubation, the tubes were microcentrifuged for 15 sec. at 10,000 rpm. The supernatant was aspirated and the pellet was suspended in 180 μl binding medium and layered on to 800 μl cold D-PBS containing 10% sucrose. The tubes were centrifuged for 1 min by microcentrifugation. The supernatant was aspirated carefully and the pellet was collected by cutting the tip of the tube and the radioactivity present in the pellets was measured by a gamma counter. RESULTS Identification of MGSF Binding Proteins by the MGSF Affinity rv_;iι-τnn MGSF binding proteins have been identified with an MGSF-sepharose affinity column. When Hs294T membrane preparations were iodinated by the lactoperoxidase method, bound to MGSF-sepharose, and eluted with 100 mM HC1, followed by lyophilization and reducing SDS- polyacrylamide gel electro-phoresis prior to autoradiography. The results indicate 125 I-membrane proteins of >200,000 and -65,000 molecular weight bind MGSF-sepharose suggesting that these two proteins are the major MGSF binding proteins. Proteins in these molecular weight ranges had a slight affinity for sepharose alone, as is shown in Fig. 1. Binding of 125 I-MGSF to Cells Binding studies with natural 125 I-MGSF and Hs294T cells consistently produced low levels of binding. Tests with several different binding buffer preparations by altering [Ca++], [Mg++] , [BSA] , [ovalbumin] , pH, ionic strength, and other parameters indicated that optimal binding could be achieved ith a binding buffer of PBS, ph 7.2, containing 10 mg/ml BSA and HEPES (30 mM) . Binding assays performed on sub-confluent cultures of Hs294T cells in 24 well plates for 2 hours at 25° C typically specifically bound about 0.74 fmoles of MGSF per 10 6 cells (-445 receptors per cell) . Prewashing cultures 5 times prior to binding helped remove endogenous MGSF and enhanced 125 I-MGSF binding. Suramin pretreatment followed by 5 washes did not improve over the prewash treatment alone. Half maximal binding was achieved in the presence of -10 ng unlabelled natural MGSF. When 125 I-rMGSF binding studies were performed on suspensions of Hs294T cells using the protocol for IL-8 bindings studies, the results indicated approximately 480 high affinity receptors per cell, the calculated Kd was 0.5 x 10 "10 M, as compared to the Kd of IL-8 for the neutrophil IL-8 receptor of 8.0 x 10 "10 M. One difficulty encountered in the binding studies using rMGSF is that concentrations of cold rMGSF of 200 ng/ml inhibited binding to the same extent as 1 μg/ml or 2 μg/ l. this is at least partially the result of the tendency of MGSF to form dimers and tetrameres at neutral pH so that the concentration of free monomer available for binding does not actually increase as the unlabelled MGSF increases to 2 μg/ml. Additionally, there could be a very large number of low affinity and perhaps less specific binding sites on the cells studies (Fig. 2b) . When 125 I-MGSF was tested for ability to bind to a number of cell types, a low level of binding was obtained with melanoma, polymorphonuclear leukocytes (PMN's), and Balb/c 3T3 fibroblasts, lymphocytes, nevus cells, lung carcinoma, prostatic carcinoma, NRK49-F and glioblastoma cells, while other cell types tested exhibited a very low binding capacity for 125 I-MGSF (neuroblastoma and FS-4 human fibroblasts) (Table 1) . Bioresponse to MGSF Numerous cell lines were examined for ability to bind 125 I-MGSF and exhibit a bioresponse to MGSF. Bioresponse to MGSF was assessed by either cell number or 3 H-thymidine assay. 3 H-thymidine incorporation assays were performed on serum depleted confluent cultures. Cell lines which exhibited a strong MGSF response were MeWo LCI, A427 lung carcinoma, nevus 106, Calu-6 adenocarcinoma of the lung, PC3 prostatic carcinoma, NRK-49F normal rat kidney, FS4 human fibroblasts. One human fibroblast culture did not respond and the A172 glioblastoma cell line did not respond. Nevus 106, Hs294T melanoma and Balb/c 3T3 cells gave a modest response to exogenous MGSF (Table 1) . Competition of rMGSF with 125 I-IL-8 Neutrophil Binding Assay The observation that MGSF binds to human neutrophils and lymphocytes suggested that there may be some relationship between MGSF and other members of the B-TG family which are chemotactic for neutrophils. Consequently, the ability of MGSF to compete with 125 I-IL- 8 for its binding sites on the neutrophil was examined. 125 I-IL-8 specifically binds to human neutrophils and 100 ng unlabelled IL-8 displaces approximately 50% of the labelled 125 I-IL-8 from these cells. rMGSF competed with 125 I-IL-8 for binding to its receptor with 50% competition at 500 ng/ml. This represents a concentration ~5 times that of IL-8 for 50% inhibition of binding of 125 I-IL-8 to this receptor (Fig. 3) . Neutrophil Chemotactic Activity in rMGSF Since IL-8 shows in vitro neutrophil activity, the possible chemotactic activity of rMGSF for human neutrophils was tested. Concentrations of as little as 0.5 ng/ml of MGSF resulted in a chemotactic index of 3.0 while the peak response was at 50 ng/ml with a chemotactic index of 17.6. There was a decline in the chemotactic index with 500 ng/ml of MGSF. For comparison 10 "7 M fMLP resulted in an index of 21.2. This was comparable to the 50 ng/ml concentration of MGSF (Table 2) . Thus, the dose dependent potency of neutrophil chemotactic activity of MGSF is essentially equal to recombinant IL-8 (Larsen et al, supra) . MGSF Bioactivity in IL-8 Preparations Based upon the demonstration that rMGSF binds to the IL-8 receptor and exerts chemotactic activity, it was desired to be determined whether IL-8 might also be capable of eliciting an MGSF biological response in the Hs294T melanoma cultures using the MGSF bioassay as described in Bordoni, et al, J. Cell Biochem. 39:241-248, 1989. Therefore, the ability of rIL-8 to stimulate 3 H- thymidine incorporation into DNA in Hs294T cells was examined. IL-8 preparations exhibit MGSF bioactivity at concentrations of 60 ng/ml and 6 ng/ml, but 0.6 ng/ml and 0.06 ng/ml were inactive. The percentage of stimulation was equivalent to that of 0.6 ng/ml preparations of rMGSF, yielding a modest stimulation of 3H-thymidine incorporation into DNA in low density serum free Hs294T melanoma cultures (Table 3) . In short, the results presented herein clearly demonstrate the neutrophil chemotactic activity of MGSF. This newly established properly of MGSF now allows for the first time to use MGSF as an inducer of neutrophil activity which may be desired in such conditions as Neutropenia or in those conditions which result from neutrophil dysfunction. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Table 1 Relativvee 112255 II--MGSA MGSA Bioresponse Cell Type Binding as %Hs294T Assay 1 Hs294T melanoma 100% *159% (1.8 ng/ml) MeWo LCI melanoma 45% **221% (.15 ng/ml) SK-N-Mσ neuroblastoma 38% ND A172 glioblastoma 273% **125% (6ng/ml) PC3 prostatic carcinoma 56% *260% (1.8 ng/ml) A427 lung carcinoma 139% **188% (.6 ng/ml) Calu 6 Adenocarcinoma 170% *263% (1.8 ng/ml) (probable lung) Nevus 106 62% *157% (.6 ng/ml) NRK-49F rat fibroblasts 277% *257% (0.18 ng/nl) Balb c/3T3 mouse fibroblasts 204% **150% (6ng/ml) FS-4 human fibroblasts 22% **333% (6ng/ml) LeSans human fibroblast ND no response lymphocytes-human PB 68% ND granulocytes-human PB 90% ND ** bioresponse data from cell number experiment * bioresponse data from 3 H-thymidine incorporation assay. 1 Bioassay data shown are as percent of binding buffer control for the concentration yielding the maximal response. Table 2 Chemotaxis Assay Migrated Cell Number/High Power Field (40X10) Chemotatic* =6 ue s s own ♦Chemotactic Index= Migrated cell number in the presence of chemoattractant. Migrated cell number for media alone. Table 3 Effect of IL-8 and rMGSA on Hs294T 3 H-Thymidine Incorporation % BSA Control 3 179% 173% 133% 112% 134% 173% 133% 132% 1 Samples are initially aliquoted with 50 ug BSA so that as each sample is diluted for bioassay, the BSA concentration diminishes. Therefore, the data are normalized to a dilutions of BSA aliquots equivalent to the BSA in the growth factor samples.

Claims: WHAT IS CLAIMED IS: 1. A method for stimulating neutrophil activity, comprising exposing leukocytes to an effective amount of MGSF to produce increased neutrophil number and activity. 2. A method for combating immunodeficiency state, comprising exposing leukocytes to MGSF to produce increased neutrophil number and activity resulting in enhanced immune function.

Inventors: MATSUSHIMA KOUJI, SAMANTA AJOY K, ZACHARIAE CLAUS O, MATSUSHIMA, KOUJI, SAMANTA, AJOY, K., ZACHARIAE, CLAUS, O.
Applicants: US COMMERCE, THE UNITED STATES OF AMERICA, AS REPRESENTED BY TH
Application year: 1991
Main classification: A61K37
IPC codes: A61K38, A61P7, A61P37, C07K14