Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor: effect on stability and biological activity.

Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2 and t-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2 oxidation of Met122 is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127 and Met122 is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138 is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127 is less accessible to solvent and Met122 is near the hydrophobic core. Oxidation at both Met127 and Met122 results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127 or at Met138 are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retain in vitro biological activity following oxidation.

The majority of stem cell factor exists as monomer under physiological conditions. Implications for dimerization mediating biological activity.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

Epitope mapping and immunoneutralization of recombinant human stem-cell factor.

The epitope regions of three anti-[stem-cell factor (SCF)]g have been mapped by characterization of immunoreactivities against truncated forms of SCF in immunoblots and against synthetic peptides in solution-phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61-95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31. The ability of the anti-SCF Ig to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured, mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61-65 and 91-95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF.

Expression patterns of immediate early transcription factors in human non-small cell lung cancer. The Lung Cancer Study Group.

In 1995, there will be 172,000 new cases of lung cancer diagnosed and 153,000 deaths from this disease in the United States. While the pathogenesis of the disease process is poorly understood, a growing body of evidence suggests that abnormalities in cellular regulatory genes may play an important role in the induction, maintenance and/or progression of some tumor types. These genes include both growth promoting oncogenes as well as growth inhibitory or suppressor genes. Included among these genetic sequences are several cellular transcription factors. A group of these factors including c-jun, c-fos and EGR1 are members of a class of genes known as immediate early genes whose expression are inducible by a variety of stimuli including mitogenic and differentiation inducing growth factors, indicating a potential important role for these genes in normal growth processes. Since these genes are involved in early regulation of cellular growth properties and at least two (c-jun and c-fos) can act as oncogenes, we wished to determine whether their expression levels were altered in human non-small cell lung cancers (NSCLC) compared to normal lung tissue. To address this, Northern blot analyses were performed using c-fos, c-jun and EGR1 probes on RNA extracted from 101 NSCLC tumor specimens and adjacent uninvolved lung tissue. Analysis of this cohort revealed that 72% of the normal tissues demonstrate significantly greater expression of these transcription factors as compared to adjacent malignant tissue. Moreover, this expression pattern appeared to be coordinate for all three genes in the majority of cases. This differential expression pattern was confirmed at the protein level using an immunohistochemical approach with antibodies directed against the c-jun, c-fos and EGR1 gene products. Southern blot analyses demonstrated no gross alterations of these sequences at the DNA level, indicating that the observed differential expression pattern was not due to gross structural changes in the genes. These data suggest that down-regulation of these genes may be involved in the pathogenesis of lung cancer.

Antibodies to canine granulocyte colony-stimulating factor induce persistent neutropenia.

A severe persistent neutropenia developed in a rabbit that was injected intradermally with 120, 60, 60, and 120 micrograms of recombinant canine granulocyte colony-stimulating factor (cG-CSF) on days 1, 22, 31, and 44, respectively. The neutropenia was present from day 44 to day 205. The nadir of the neutropenia (60 cells/microliters) occurred in conjunction with peak antibody titer (640,000) to cG-CSF on day 58. The immune antiserum from this rabbit reacted positively for cG-CSF on Western blot analysis. The immune antiserum also neutralized the activity of cG-CSF. On day 160, examination of the bone marrow showed marked granulocytic hypoplasia and mild erythroid hyperplasia. On day 205, the rabbit was still neutropenic (430 cells/microliters), even though the last injection of cG-CSF was given 161 previously. Necropsy on day 205 showed that there was still mild granulocytic hypoplasia with mild erythroid hyperplasia. Because of the lack of any inflammatory foci found at necropsy and the granulocytic hypoplasia, it was thought that the neutropenia was most likely due to decreased production and was not a consumptive process. It is hypothesized that the antibody that was produced to cG-CSF neutralized the effect of endogenous rabbit granulocyte colony-stimulating factor and prevented the normal proliferation and maturation of the rabbit neutrophils.

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed.

Oh-eda et al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 [J. Biol. Chem. (1990) 265, 11,432-11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased granulopoiesis after sequential administration of transforming growth factor-beta 1 and granulocyte-macrophage colony-stimulating factor.

Transforming growth factor-beta 1 (TGF-beta 1) is an inhibitor of the growth and differentiation of immature hematopoietic progenitors in vitro; however, we have demonstrated that TGF-beta 1 can promote granulopoiesis in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. We therefore examined the effect of the combined administration of TGF-beta 1 and GM-CSF in vivo. First, TGF-beta 1 enhanced the specific binding of GM-CSF (2.0-fold) on bone marrow cells, reaching a maximum 40 hours after injection, while the specific binding of interleukin-3 (IL-3) was unaffected. Using GM-CSF-specific binding to determine the optimal regimen for cytokine administration in vivo, we found that the administration of TGF-beta 1 and GM-CSF in sequence increased myelopoiesis. Total numbers of colony-forming units-granulocyte/macrophage (CFU-GM) and myeloblasts per femur were increased above the level obtained with the simultaneous injection of TGF-beta 1 plus GM-CSF, GM-CSF alone or TGF-beta 1 alone. Further, the sequential administration of TGF-beta 1 and GM-CSF resulted in enhanced numbers of mature granulocytes in both the bone marrow and peripheral blood. In contrast, the sequential combination of TGF-beta 1 and GM-CSF did not enhance the numbers or increase the recovery of erythroid cells in the bone marrow. These results show that TGF-beta 1 in vivo as in vitro has a multifunctional effect on bone marrow progenitors, and by using an optimal combination of TGF-beta 1 and GM-CSF in vivo, one can selectively increase both the central and peripheral granulopoietic compartments.

Glycosidase digestion, electrophoresis and chromatographic analysis of recombinant human granulocyte colony-stimulating factor glycoforms produced in Chinese hamster ovary cells.

Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.

Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin.

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Comparative analysis of the effects of recombinant cytokines on the growth and differentiation of ML-1, a human myelogenous leukemic cell line.

We have investigated the effect of a number of cytokines on the human acute myelomonocytic leukemic cell line, ML-1. The differentiation inducing effects of interleukins (IL-1 beta, IL-3 and IL-6), colony stimulating factors (GCSF and GMCSF), TNF, LIF and IFNg, were studied either individually or in combination. Criteria for monocytic differentiation were as follows: an increase in the percentage of cells reducing nitroblue tetrazolium (NBT) salt, an increase in the alkaline phosphatase activity as well as the appearance of macrophagic phenotype. Among all the cytokines tested, only TNF was found to induce differentiation and to inhibit growth of ML-1 cells. IL-3, IL-6, interferon gamma, GCSF and to a smaller extent IL-1 beta and GMCSF synergized the differentiation inducing activity of TNF.

Effect of recombinant canine granulocyte colony-stimulating factor on peripheral blood neutrophil counts in normal cats.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously at a dosage of 5 micrograms/kg/day to five healthy, young adult cats for 42 days. Mean neutrophil counts +/- standard deviation increased significantly (P < 0.001) from 10,966/microL +/- 2324 to 30,688/microL +/- 5296 within 24 hours after administration of the first dosage of rcG-CSF. Mean neutrophil counts reached 52,978/microL +/- 11,207 on day 6, representing a second significant increase (P < 0.01) over the previous 5 days. Mean neutrophil counts continued to increase, reaching 66,994/microL +/- 12,419 on day 14, then remaining within a range of 66,994 to 87,839/microL throughout the remainder of the study. The maximum mean neutrophil count was 87,839/microL +/- 8,695 on day 42. Neutrophil counts remained high until the administration of recombinant canine granulocyte colony-stimulating factor was discontinued 42 days after initiation of therapy. Once the rcG-CSF administration was discontinued, neutrophil counts returned to pretreatment values within 5 days. There were no significant changes in numbers of any of the other cell lines. There was no clinically significant toxicosis associated with the administration of rcG-CSF.

Neutrophil function in normal and Chediak-Higashi syndrome cats following administration of recombinant canine granulocyte colony-stimulating factor.

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined. CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions. Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3. The drug was specific for neutrophils as determined by differential cell counts. Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo. Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E. coli. At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis. CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period. We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.

Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping.

Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.

Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases.

The balance between levels of metalloproteinases and their corresponding inhibitors is a critical factor in tumor invasion and metastasis. Down-regulation of the activity of these proteases was achieved by transfection of invasive and metastatic rat cells with the complementary DNA for metalloproteinase inhibitor/tissue inhibitor of metalloproteinase 2 (MI/TIMP-2), a novel inhibitor of metalloproteinases recently described. (Y. A. DeClerck et al., J. Biol. Chem., 264: 17445-17453, 1989; W. G. Stetler-Stevenson et al., J. Biol. Chem., 264: 17374-17378, 1989). Secretion of functional MI/TIMP-2 protein in stably transfected cells resulted in a marked decrease in metalloproteinase activity. Partial suppression of the formation of lung colonies after i.v. injection in nude mice was observed in a transfected clone expressing high levels of MI/TIMP-2. Production of MI/TIMP-2 in four clones markedly reduced tumor growth rate in vivo after s.c. injection and completely suppressed local tissue invasion. Thus, down-regulation of metalloproteinase activity has a striking effect on local invasion and partially suppresses hematogenous metastasis.

Use of recombinant canine granulocyte colony-stimulating factor to decrease myelosuppression associated with the administration of mitoxantrone in the dog.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (less than 3,000/uL) for 2 days in the dogs that received rcG-CSF, and for 5 days in the dogs that received only mitoxantrone. Four of five dogs not treated with rcG-CSF and none of those receiving rcG-CSF developed serious neutropenia (less than 1,500/uL). The neutrophil counts were significantly (P less than 0.05) higher in the rcG-CSF treated dogs at all time points except before the administration of the colony-stimulating factor, and the sixth day after the mitoxantrone was administered. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.

Granulocyte colony-stimulating factor effects on lymphocytes and immunoglobulin concentrations in periparturient cows.

Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5 micrograms/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows.