CA2075191A1 - Method for treating immunodeficiency or neutropenia - Google Patents

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

- - 207al91 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 (MG5F).
BACKGROUND OF THE INVENTION
Melanoma growth stimulatory ~actor (MGSF) is a growth related protein from B-thromboglobulin (B-TG) superfamily. Although ~GSF has a number o~ properties similar to interleukin-8 (IL-8), another member o the B-TG family, MGSF has not heretofore been demonstrated to possess neutrophil chemotactic activity.
SUMMARY OF THE INVENTION
It is, therefore, an object o~ the present invention to demonstrate neutrophil chemotactic activity of MGSF.
It is another o~ject of the pressnt 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.
~IEF ~EscRIpTl~N QF THE DRA INGS
These and other objects, features and many of the attendant advantages of t~e 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 prote~n by ligand affinity binding. 125I-membrane proteins from Hs294T melanoma cells were allowed to bind to rMGSA-:. ,~ 1 . . .- , ~: , ~ . . . . : . . . : . ':
.. .., , .
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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) 125I-Hs294T membrane preparation eluted from 4-MGSA-sepharose.
Lane b) l25I-Hs294T membrane preparation eluted from sepharose alone.
Lane c) 125I-Hs294T membrane preparation.
Figure 2a shows MG5F Binding Curve. Human Hs294T
melanoma cells were grown to confluency and incubated in SF culture medium for 48 hours. Cultures were then incubated with l25I-MGSA and varying concentrations of unlabeled MGSA for 2 hours at 25-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 12sI-rMGSA and unlabeled rMGSF as described in Materials and Methods. Cells were wa~hed once, pelleted through a sucrose cushion and bound radioactivity was counted on a ga~ma counter. A
Scatchard plot of the binding data is shown.
Figure 3 shows MGSF/IL-8 Competitive ~indings Curve.
Human neutrophils (2xlO6) were suspended in binding medium and incubated with lZ5I-IL-8 wi~ or wi~hout varying concentrations of unlabeled MGSF or IL-8 for 2 hours at 4-C. Cells were washed once, pelleted through a sucrose cu~hion, and bound radioactivity was counted nn a gamma - counter.

207~

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 tachnical 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 prQsent invention, the pre~erred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned o~herwise, the techniques employed or contemplated herein are standard methodologie~ well known to one o~ ordinary skill in the art. The material~, methods and examples 20 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 ~tudies described herein, either r~GSF or natural ~GSF was employed as Pound convenient or as noted herein. ~he specifics of the methods are now described.
~A~ a~ ODS
P~epa~at~on o~_~lasma Membrane-enriçhed Fract~on _~om ~s294T Melanoma Cel~s 3~ Confluent P-150 dishes (5 per preparation~ of Hs294T
cells were maintained on serum-~ree medium for a minimum o~ 48 hours. After washing with serum-free medium, the , . . , - . ,~
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, ' ' ;' ~ , ~ , ,,, ' ; ' ' ' ,_ ~ 4 cells were removed by scraping and sedimented by centrifugation. The weights of the cell pellets were determined and the cells were resuspended (l:lO w/v) in a hypotonic buf~er solution (50 mM mannitol, 5mM Hepes, lOug/ml leupeptin, ~% aprotinin pH 7.4). The cell suspension was passed through an 18 gauye needle, l M
CaCl2 was added to ~hi5 suspension to a final concentration of lO mM and the suspension was maintained on ice for lO 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 l minute at 4~C. The supernatant was ultracentrifuged at 105,000 xg for 90 minutes at 4'C producing a fine translucent pellet that was resuspended in hypotonic bu~fer and stored at -80C.
Iodination of Hs2~4T Nembr~nes A suspension of Hs294T membranes (10.8: ug) was microfuged for 15 min. at 4C. The pellet was resuspended in 2.5 ~l 0.5 M sodium phosphate. pH 7.2. To the suspended membranes, 20 ~l of lactoperoxidase, lO ~l o~ glucose oxidase, and lO~l 125I-Na (l mCi) were added.
The reaction was initiated by the addition of 20 ~l B-D-gluco~e (l8 mg/ml H20 freshly prepared) followed by incubation of the reagents for l5 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-lO0 pH 7.2. This solutio~ was trans~erred to a PD-lO column that was previously washed with 50 ml H2O, 2 ml 0.05 M sodium phosphate, 0.1% Triton X-lO0, 20 mg/ml bovine serum albumin pH 7.2 followed by 30 ml 0.05 M sodium phosphate, 0.1% Triton X-lO0 pH 7.2.
Material w~s eluted using 0.05 M sodium phosphate, 0.1%

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W O 91/11197 P ~ /US9~/00572 207~191 Triton X-100 pH 7.2. Fractions (2 ml) were collected into tubes containing 1 ml o.05 M sodium phosphate, 0.1%
Triton X-100, 20 mg/ml bovine serum albumin pH 7.2. A 5 ~1 aliquot of each fraction was counted in a gamma counter, then diluted with 2X laemmli electrophoresis sample buffer for visualization by standard SDS-P~GE and autoradiographic analyses, Puri$ication Q~ NGSF
Natural MGSF was purified ~rom 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 puri~ied from conditioned media from chinese hamster ovarian (C~0) cells stably transfected with the MGSF gene placed under the control`
15 of the cytomegalovirus promoter as described by Balentien ;~
et al, supra.
PreParation of Sepharose-~ound Recombinant MGSF ~' Cyanogen bromide activated sepharose (O.1 g) was incubated overnight (about 12-16 hrs) with 15 ml lmM HCl.
The swollen resin was washed three time~ with 2.5ml aliquots of 1 ~M HCl ~hen suspended in 375 ~1 of coupling buf~er (98.4 g sodium bicarbonate, 29~2 g sodium chloride per iiter water, pH 8.3~. Recombinant MGSF (5 ~g per 375 ~1 coupling bu~fer) was added to the suspended resin and agitated overnight at 4-C. The resin was pelleted by centrifugation at 500 rpm in a Beckman ~J-6 centrifuge.
The supernatant was removed and the resin was washed two times with coupling bu~fer. The resin was incubated overnight at 4-C with 1 ml of monoethanolamine. A~ter removing monoethanolamine, the resin was washed with ac~tate bu~fer (8.2 g sodium acetate, 58.4 g sod~um chloride per liter water, pH 4.0) ~ollowed by Tris bu~fer (6.075 g Tris, 11.6 g sodium chloride per liter water, pH
8.0). The acetate ~u~fer - Tris buffer cycle was repeated three times. The resin was stored in o.05 M
sodium pho~phate buffer, pH 7.2 ~ LJ~L_ o~ l2sI-La~ d ~s22~ ~nes to r~GSF-Seph~rose rMGSF-sepharose was suspended in 30 ~1 of binding buffer (0.8g ~odium chloride, 0.115 g dibasic sodium phosphate, 0.02 g potassium chloride, 0.02 g monobasic potassium phosphate, 0.01 g magnesium chloride, o.o1 g calcium chloride, 1.O g bo~ine serum albumin, 1.0 mg leupeptin, and 10,000 kallikrein inhibitory units of aprotinin per 100 ml water, pH 7.2). 125I-labelled Hs294T
membranes (35 ~1, 5,000,000 cpm) were added to rMGSA-sepharose and a control sepharose resin. These wereincubated at 4-C with agitation for 4 hrs. The resin was pelleted by centrifugation and the supern~tant 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 HCl for 15 min. A~ter pelleting the resin the supernatant was removed, the elutio~ was repeated and the aliquots were combined and frozen. The solution was dried by speed vac and the material was redissolved in 20 ~1 4 mM HC1. Laemmli 2X electrophoresis sample buffer (40 ~1) was added, the solutions were boiled for 5 min., microfuged for 2 min., and the supernatants were electrophoresed in a 7.5% polyacrylamide gel. After staining with Coomassie blue R-250, the gel was dried and exposed to Kodak XAR5 film for ~isualizatio~ of the radiolabelled proteins.

: : ,:. ~j,,, :, .. : ,, j ~ .: .,,, : , WO91/1~197 PCT/US91/00572 2~7~191 Competitive Bindinas Assays ~ MGSF was iodinated by either Bolton-Hunter or the chloramine-T procedure ~Bolton et al, ~iochem J. 133:529-539, 1973: Hunter et al, Nature (London) 194, ~95 1962).
Studies of l2sI-MGSF (specific activity 6.7Xl05 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 H~PES + 1% .
BSA, p~ 7.2. After the appropriate length of time, the binding buffer was aspirated from the monolayers and the cells were washed ~X with ice cold binding buffer. The monolayers were then treated with 1 ml of lysis buffer (o.5 g ~DS, 0.121 g Tris, 0.037 g E~TA/lOO ml H2O) at room temperature for 15 min. The lysis bu~fer was transferred to 12 x 7.5 mm tubes. The wells were rinsed with a second aliquot (0.5 ml) of lysis buf~er. The aliquots were combined and the amount of l2sI-MGsF found was determined in a gamma counter. Non-specific binding was determined in the presence of lOO fold excess crude MGSF.
Studies of the binding of l25I-MGSF to several normal and malignant cell lines were per~ormed on suspension cultures ~1XlO6 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 centri~ugation 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~ .
M~SF ~ioresponse ~ssay The bioresponse elicited by exogenous MGSF in various cells lines was determined by 3H-thymidine incorporation : . , ., : .
- .
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wos~/11197 PCT/US~1/00572 assay or in a si~-day cell number assay. For tritiated thymidine incorporation assays 104 cells were plated into 96 well paltes (Falcon) în DME~ medium (Gibco) containing 1~ fetal bovine serum and incubated overnight at 37C in a water jacketed C02 incubator. The following morning aliquots of ~GSF or fetal bovine serum were added, the incubation was continued for an additional 18 hours, and then 5 ~Ci of 3H-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 lOo ~l phosphate buffered saline and fixed with 5%
trichloroacetic acid (TCA) for 30 min on ice. The TCA
- was aspirated and 200 ~l of 100% methanol was added to each well. After 30 min the methanol was aspirated and cells were solubilized in lOO ~l of 0.3N NaOH for l hr at 37-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-~ree medium.
Twenty-four hours later, the medium was aspirated and replaced with serum-free medium with and without aliquots of ~GSF. A background count was done on well containing only serum-~rQQ medium. On thQ third and sixth days a~ter growth factor additions, cell number was determined. Tho~e cultures that were not counted on day three were ~ed at that ~$me.
Chemot~xls_AssaY
~he nautrophil chemotaxis assay was performed as describQd by Larsen et al., Science 243:1464-1466, 1989 and Falk et al, J. Immunol. Method. 33:239-247, 1980, .
2075~91 except that rMGSF wa~ used.
5I-IL-8 Competitive Bindin~ Assav Recombinant IL-8 has been expressed in E. coli and purified to homogeneity with specific activity of 2XlO~/mg (Furuta et al, J. Biochem. 106:436-441, 1989).
Recombinant IL-8 was labelled with l25I 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 125I-IL-8 10 (1. 0 X 107 cpm/~g) to human neutrophils was examined. In standard binding assays, 2xl06 cells were incubated in duplicate with 4 ng 125I-IL-8 (40,000 cpm) in RPMI 1640 medium containing lO mg/ml BSA and 20 mM HEPES bu~fer pH
7.2 in a total volume of 200 ~l. Samples were mixed and incubated at 4-C for 2 hours. After incubation, the tubes were microcentrifuged for 15 sec. at lO,000 rpm.
The supernatant was aspirated and the pellet was suspended in 180 ~l binding medium and layered on to 800 ~l cold D-PBS containing 10% sucrose. ~he tubes were centrifuged for l 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 Bindinq Proteins by the MGSF
Affinity Column MGSF binding proteins have been iden~ified with an MGSF-sepharose affinity column. When Hs294T-~embrane preparations were iodinated by the lactoperoxidase ~ethod, bound to MGSF-sepharose, and eluted with lO0 mM
HCl, ~ollowed by lyophilization and reducing SDS--; . ~ . :

- ; WO91/11197 PCT/US91/00~72 polyacrylamide gel electro-phoresis prior to autoradiography. The results indicate 12sI-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. l.
Bindina o~ 125I-MGSF to Cells Binding ~tudies with natural l2sI-MGSF and Hs294T cells consistently produced low levels of binding. Tests with several different binding buffer preparations by altering ~Ca++], [~g++], [BSA], ~ovalbumin], pH, ionic strength, and other parameters indicated that optimal binding could be achieved with a binding buffer of Pss, ph 7.2, containing l0 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 lo6 cells ~-445 receptors per cell). Prewashing cultures 5 times prior to binding helped remove endogenous MGSF and enhanced l25I-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 l0 ng unlab~lled natural MGSF.
When ~25I-rMGSF binding studies were performed on suspensions o~ 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 l01M, as compared to the Kd of IL-8 for the 30 neutrophil IL-8 receptor of 8.0 x l01M. One difficulty enaountered in the binding studies using rMGSF is that concentrations of cold rMGSF of 200 ng/ml inhibited :`' . ' '' ' .~ ~ .: . : ~ " ' ; . . , WO91/11197 pcT/uss1ioo572 207~191 11 .... ,~
binding to the same extent as 1 ~g/ml or 2 ~g/ml. 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 ~g/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 lZ5I-MGSF was tested ~or 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, luny carcinoma, prostatic carcinoma, NRK49-F and glioblastoma cells, while other cell types tested exhibited a very low binding capacity for 125I-MGSF (neuroblastoma and FS-4 human fibroblasts) (Table 1).
~iores~onse to MGSF
Numerous cell lines were examined ~or ability to bind 125I-MGSF and exhibit a bioresponse to MGSF. Bioresponse to MGSF was assessed by either cell number or 3H-thymidine assay. 3H-thymidine incorporation assays were performed on serum depleted confluent cultures. Cell lines which exhibited a strong MGSF response were MeWo LCl, A~27 lung carcinoma, nevus 106, Calu-6 adenocarcinoma of the lung, PC3 prostatic carcinoma, NRK-49F normal rat kidney, FS4 human ~ibroblasts. One human fibroblast culture did not respond and the Al72 glioblastoma cell line did not respond. Nevus 106, Hs294T melanoma and ~alb/c 3T3 cells gave a modest response to exogenous MGSF (Table 1).

... . . . .. .. ....

WO9~/11197 ~ ~9~ PCT/US91/00572 Competition of rMGSF _with 125I-I~-8 Neutrophil Bindin~
~ssay The observation that ~GSF binds to human neutrophils and lymphocytes ~uggested 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 125I-IL-8 f or its binding ~ites on the neutrophil was examined.
125I-IL-8 specifically binds to human neutrophils and lOO
ng unlabelled IL-8 displaces approximately 50% of the labelled 125I-IL-8 from these cells. rMGSF competed with 1Z5I-IL-8 for binding to its receptor with 50% competition at 500 ng/ml. ~his represents a concentration -5 times that of IL-8 for 50% inhibition of binding of i25I-IL-8 to this receptor (Fig. 3).
Neutro~hil Chemotactic Activitv 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 ch,emotactic index of 17.6. There was a decline in the chemotactic index with 500 ng/ml of MGSF. For comparison 10'7M 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 o~ ~GSF is essentially equal to recombinant IL-8 (Larsen et al, supra~.
~SF_Bioactivity 'n 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 -WO 91tlll97 PCT/US91/00572 2~7~jl91 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. 39:241-24~, -1989. Therefore, the ability of rIL-8 to stimulate 3H-thymidine incorporation into DNA in Hs294T calls was examined. IL-8 preparations exhibit MGSF bioactivi~y at concentrations of 60 ng/~l 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 cIearly demonstrate the neutrophil chemotactic activity o~ 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 dys~unction.
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.

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Table l Relative 125 I-MGSA MGSA Bioresponse Cell Type Bindina as ~Hs294T Assayl Hs294T melanoma 100% *159% (l.8 ng/ml) MeWo LCl melanoma 45% **221% (.15 ng/ml) SK-N-Mc neuroblastom~38% ND
Al72 glioblastoma 2~3% **125% (6ng/ml) PC3 prostatic carcinoma 56% *260% (1.8 ng/ml) A427 lung carcinoma139% **188% (.6 ng/ml) Calu 6 Adenocarcinoma 170% *263~ (l.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 ~0 lymphocytes-human PB 68% ND
granulocytes-human PB 90% ND
** bioresponse data from cell number experiment * bioresponse data from 3H-thymidine incorporation assay.

Bioassay data shown are as percent of binding bu~fer control for the concentration yielding the maximal response.

2075~1 .Table 2 Chemotaxis Assay Migrated Cell Nu~ber/High Power Field (40XlO) Chemotatic*
~GSA Contraction ~n=6. mean yalue ls shown) Index 0 5.8 0.05 ng/ml 6,0 l.l 0.5 ng/ml 17.5 3.0 5.0 ng/ml 48.3 8.3 15 50.0 ng/ml lOl.8 17.6 500 ng/ml 86.5 14.9 fMLPlO7M 123.0 21.2 *Chemotactic Index=
Miqrated cell number in the presence of chemoattractant.
Migrated cell number for media alone.

: : ;
W~91/11197 ~ PCT/US91/00572 :`

Table 3 Effect of IL-8 and r~GSA
on Hs294T 3H-Thymidine Incorporation IL-8 c~m~S.D. ~ BSA_Control 1) 60 ng/ml 2136+174 179%
2)` 6 ng/ml 2167~407 173%
10 3) 0.6 ng/ml 2204+481 133% ~:
4) 0.06 ng/ml 1761+231 112%
rMGSA
1) 6 ng.ml 1607+415 134%
2) o6 ng/ml 2164+565 173%
15 3) .06 ng.ml 2192+173 133%
4) .006 ng/ml 2078+420 132%
BSA dilutions 1) 12.5 ug/ml 1209~328 2) 1.25 ug/ml 1253+203 20 3) .125 ug/ml 1655+454 4) 0.125 ug/ml 1578+586 .
~ 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 o~ BSA aliquots equivalent to the ~SA in the growth factor samples.

Claims (2)

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.