IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 13(3), 379-393 (1991)

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

THE ANTIFOLATE 10-DEABAAMINOPTERIN INHIBITS NEUTROPHIL CHEMOTAXIS

AND SUPEROXIDE GENERATION

Georg F. Weber.’,

M. G. Nair, and Joe M. McCord

College of Medicine, Department of Biochemistry University of South Alabama Mobile, Al. 36688 Phone: (205) 460-6857

ABSTRACT The effect of the dihydrofolate reductase inhibitor 10deazaaminopterin on several neutrophil functions was tested in vitro. At 100 uM it inhibited chemotaxis by 50% and reduced the generation of superoxide by 30%. It had no influence on phagocytosis and did not significantly change the secretion of B-Dglucuronidase, a marker enzyme of degranulation. After preincubation of white cells with various concentrations of 10deazaaminopterin, followed by resuspension in drug-free medium, no inhibition of chemotaxis or superoxide generation was seen. Therefore, the effects on chemotaxis and NADPH oxidase appear to be reversible and not due to metabolic transformation of the dihydrofolate reductase inhibitor.

INTRODUCTION lo-deazaaminopterin (Figure 1) is an inhibitor of dihydrofolate reductase. It was developed in an effort to improve



Present address: J-525 , Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, phone (617) 732-3108 319

Copyright 0 1991 by Marcel Dekker, Inc.

380

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

OH

CH,HZN

-

H

WEBER, N A I R , AND MC CORD

O

COOH

0

FOOH

1

COOH

Methotrexate 0

CH,H2N

C

-H

C - N -C

N

H

2

YOOH

aC-N-C-H I'

10-Deazaarninopterin

1COOH

FIG. 1: Chemical structures of folic acid, methotrexate, and 10deazaaminopterin (10-DAM)

the pharmacological properties of methotrexate and was found to be superior in inhibiting the growth of Streptococcus faecium or Lactobacillus casei, and in antitumor activity in vivo [6,20]. Methotrexate has been used for the treatment of rheumatoid arthritis, based on the rationale that it is effective in suppressing primary humoral and cellular immune responses and can diminish ongoing cellular immune responses [9]. Later, it was shown that there is a direct inhibitory effect on neutrophil chemotaxis [5,18,22,23].

In this study, the influences of 10-deazaaminopterin on the abilities of human neutrophils to respond to a chemoattractant, phagocytose, degranulate and generate superoxide are investigated.

MATERIALS Hank's balanced salt solution without phenol red and sodium bicarbonate was from Gibco and prepared as directed. Ficoll was from Sigma, metrizoate from Nyegaard & Co. Cytochromec (type 111, from horse heart), phorbol-12-myristate-13-acetate and zymosan A as well as p-nitrophenyl-8-D-glucuronide and cytochalasin B were purchased from Sigma. Buffer and ficoll solution were filtered through microfilters (pore-size 0.45 um) and stored at 4°C.

381

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

ANTIFOLATE 10-DEAZAAMINOPTERIN

Isolation of Leukocvtes. White blood cells were isolated from healthy volunteers, of both sexes (range of ages 21 to 61) who were not on medication and had had no infectious diseases during the preceding week. Unless otherwise indicated, the results present averages of the data from 10 donors (mean value, standard error of the mean value). Blood was drawn in heparinized tubes (500 U sodium heparin per 10 ml blood). Polymorphonuclear leukocytes were isolated by sedimentation through ficoll-metrizoate as described by Jones et al. [15]. Briefly, over a layer of ficoll-metrizoate blood was decanted gently without mixing in relation 1:2. The red cells sedimented to the bottom of the tube, being separated from the upper layer of neutrophils in plasma by the ficoll-solution. The layer containing the white cells was removed, centrifuged and the erythrocytes lysed with distilled water. The cells were washed three times and resuspended in HBSS

’.

METHODS AND RESULTS M formyl-Met-Leu-Phe Chemotaxis. The chemotactic response to was determined by a microadaption of the Boyden chamber technique. A Neuro Pore 48 well micro chemotaxis chamber was used and 5 um pore-size polycarbonate filters were purchased from the Nucleopore Corp. The well above the filter contained 4x104 cells in 50 ul Hank’s balanced salt solution containing 1 mg/ml bovine serum albumin plus different concentrations of 10-DAM as indicated. To assess random migration, preliminary series of three controls were run with equal concentrations of the chemoattractant on both sides of the filter (alteration of random movement m f sem = -7.4 f 26.5%). Each experiment was performed in triplicate, at least. The chamber was incubated 30 min at 3 7 ’ C , the filter removed, stained and mounted on a slide. The number of cells per field was counted microscopically for 6 fields, and the average was considered a measure of chemotaxis. It i s known that interindividual differences are large and the employed method does not allow comparison of absolute numbers of cells migrated. Therefore, the change in the number of cells counted under the influence of 10-DAM was calculated as percent inhibition.

382

WEBER, NAIR, AND MC CORD

.-

70

3

60

v)

X

0

r

50

U

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

0

.-+5

g C -

6\”

40

30 20 10 0

50

100

150

10-Deazaaminopterin (micromolar)

FIG. 2: Inhibition of chemotaxis by 10-DAM as determined by the Boyden chamber technique after 30 min incubation at 3 7 ’ C . Each point represents the average of 10 individuals (error bars = standard error of the mean value)

Incubation of polymorphonuclear leukocytes with 10-DAM inhibited the chemotactic response in a dose-dependent manner. The response saturated at 50% inhibition, which was reached at a concentration of 80 uM, the half-maximal effect being reached at 30 uM (Figure 2). Phaaocvtosis. Phagocytotic activity was quantified by the method described by Jandl et a1 [14]. Opsonized zymosan was prepared as described by Hohn and Lehrer [12], with the modification that 10 mg zymosan in 1 ml 0.1 N NaOH was heated in boiled water and washed three times with distilled HzO before opsonization. Neutrophils (1. 5x107 cells/ml) plus 2 mg/ml opsonized zymosan were incubated for 30 min at 3 7 ° C in the presence of varying concentrations of the antifolate. A 0.1 ml portion was withdrawn and added to 0.1 ml icecold 2 mM N-ethylmaleimide solution in HBSS to stop phagocytosis. To this was added 2 ml of a mixture containing 0.25 M sucrose, 0.1 M EDTA, 5 uM colchicine, 10 mg/ml bovine serum albumin. After agitation, a 0.4 ml portion was mounted on a slide by cytocentrifugation. The slide was dried in air and the cells fixed in 9: 1 (v/v) absolute ethanol: 3 7 % methanal. The zymosan was stained with periodic acid-Schiff and the cells were counter-stained with 20 mg/ml methyl green for 5 min followed by 20 mg/ml fast green for

383

ANTIFOLATE 10-DEAZAAMINOPTERIN

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

120 I

%-

0

6\0

2oj,. 0

0

,

.

,

50

,

,

,

,

,

,

100

.

,

,

. .

150

10-Deazaaminopterin (micromolar)

FIG. 3: Influence of 10-DAM on phagocytosis of zymosan particles by neutrophils. Each point represents the average of 5 to 8 individuals (error bars = standard error of the mean value). The control value in absence of 10-DAM was 175 +- 75 particles per 50 cells

5 min. The number of zymosan particles in contact with each of 50

cells was then determined in oi-1-immersion microscopy. There was no effect of 10-deazaaminopterin on phagocytosis at concentrations between 20 uM and 160 uM; one control measurement at a higher concentration of 320 uM (114% of control) gave no hint at an influence of the drug on phagocytosis (Figure 3). Generation of Superoxide. The generation of superoxide anion during the oxygen burst, initiated by 200 ng/ml phorbol-12-myristate-13acetate (the stock solution, 1 mg/ml in dimethylsulfoxide, was diluted 100 x in HBSS prior to use), was quantified by the c y t o c h r o m e c a s s a y with 5x10’ cells/ml according to the method described by Cohen and Chovaniec [ 4 ] . The effect of 10-DAM in the given concentrations was tested. The volume of HBSS in the cuvette was reduced according to the amount of antifolate added, so as to maintain a final volume of 2 ml. Neutrophil superoxide production was inhibited in a dosedependent manner up to a maximum of 30%, which was reached at a concentration of 160 uM with a half-maximal effect at 30 uM (Figure 4)

-

384

WEBER, NAIR, AND MC CORD

5 50

z

I

3

U

2a 4 0 0

.-U Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

2

30

Q

ul .c

0

20

C

0 ..w .r

10

C .-

:

0 0

100 200 300 400 500 600

10-Deazaaminopterin

F

,

4:

(micromolar)

Inhibition of PMN superoxide generation by 10-

AM.

Each

P --.it represents the average of 10 individuals, except the point at

6' 4 0 uM, which represents only four blood samples. (control value 3 . 7 2 +- 1.36 nmol 0,-/106 cells/min; error bars = standard error of

the mean value) The open circles show the results of the experiments with a cell-free system (averages of 5 experiments)

An inhibition of superoxide production by whole cells may either be due to a direct effect on the NADPH oxidase or to an interference with one of the processes which lead to the activation of the enzyme. Superoxide scavenging properties can virtually be excluded as there was no inhibition of cytochromec reduction by xanthine oxidase. A discrimination between these two mechanisms is possible by measuring superoxide generation in a cell-free system. Therefore, 300 ul leucocyte suspension ( lx107 cells/ml) was activated with 120 ul PMA for 2 min and the reaction stopped by addition of 4 . 9 8 ml 0.2% (w/v) Triton X 100 in HBSS. The solution was sonicated for 15 s to make sure complete breakage of the neutrophil plasma membrane. After addition of 0.2 mM NADPH the samples were assayed spectrophotometrically by the cytochromec reduction method described above. Each cuvette contained the debris of 5x105 cells/ml. After determining an initial rate, 10-DAM was added to give a final concentration of either 160 uM or 320 uM and the percent inhibition was calculated. These values are represented by open circles in Figure 4 , showing that 10-DAM produced comparable inhibition of O,-production, whether in intact cells or in preactivated cell-free preparations.

ANTIFOLATE 10-DEAZAAMINOPTERIN

385

140 C 0

. z 120 0 3

c

z

0 P)

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

-0

100

80

t

O ' ' . . . . . . ~ " ' ' ~ ' ~ ' 0 50 100 150 10-Deazaaminopterin (micromolar)

FIG. 5: Influence of 10-DAM on degranulation as determined by the release of 6-D-glucuronidase. Each point represents the average of 10 experiments (error bars = standard error of the mean value). The reference value in absence of 10-DAM was 2.9 +- 0.1 nmol/hour/lO' cells

Desranulation. The release of B-glucuronidase as a marker enzyme of degranulation was measured spectrophotometrically after hydrolysis of p-nitrophenyl A-D-glucuronide [ll]. To activate the white blood cells, 2x10' PMN/ml were incubated with 0.5 ug/ml cytochalasin B and 3 mg/ml opsonized zymosan in the presence of various concentrations 10-deazaaminopterin for 30 min at 37'C. The suspension was then centrifuged at 300 g and 4 ' C for 5 min. 100 ul of the supernatant (in a reference assay 100 ul H,O) were added to 100 ul 0.1 M p-nitrophenyl R-D-glucuronide, 100 ul 0.5 M sodium acetate buffer, pH 5.0, 200 ul H,O and the solutions again incubated at 37°C for 60 min. The reaction was stopped by addition of 1 ml 50 mM NaOH and the absorbance read at 4 0 0 nm against the blank. [19] Figure 5 shows that there was no reduction of the release of A-D-glucuronidase in the presence of lO-DAM, which indicates that degranulation was not affected by the drug. Neutrophil Phvsioloav after Transient Exposure to the Druq. A s the doses found to be effective in vitro were above the therapeutic range of plasma concentrations, we incubated white blood cells with

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

386

WEBER, N A I R , AND MC CORD

various concentrations of 10-deazaaminopterin to determine whether inhibition of chemotaxis and superoxide generation might be due to metabolism of the drug. 1x10' polymorphonuclear leukocytes per ml were incubated with lo-deazaaminopterin at the indicated concentrations in Hank's balanced salt solution containing 1 g/100 ml glucose, 1000 U/ml penicillin G I 50 ug/ml streptomycin sulfate and 5% (v/v) homologous serum in siliconized glass tubes for 24 hours at 3 7 ° C in a CO, incubator [16]. The cells were centrifuged and resuspended in HBSS in order to remove those amounts of the antifolate, which had not been taken up by the neutrophils. Then, the assays for superoxide generation and chemotaxis were carried out as described above, except that the time of incubation for chemotaxis was extended to 1 h. To ensure that changes in neutrophil physiology are not veiled by varying numbers of surviving cells, the percentage of viable neutrophils after 24 h was counted microscopically (bright field and fluorescence) after addition of 2 mg/ml fluorescein diacetate in absolute ethanol to the sample in the relation 1:lO (the dye is taken up and hydrolysed to give a fluorescent product only by viable cells). Between 65% and 80% viable cells were counted in these control determinations without any deviation at increasing concentrations of 10-DAM. After 24 hours preincubation there was no statistically significant inhibition of chemotaxis or superoxide generation (data not shown). Comparison to Methotrexate. Methotrexate is the most commonly used folic acid antagonist in the therapy of chronic inflammatory diseases like rheumatoid arthritis. We therefore compared the effects of methotrexate on neutrophil physiology to those of 10deazaaminopterin. The effect of methotrexate on neutrophil chemotaxis has been reported under a variety of conditions, with different findings. Thompson and Al-Nakeeb [23] and O'Callaghan et al. [22] reported suppression of neutrophil chemotaxis in patients being treated with methotrexate, but neither study could demonstrate a suppressive effect when normal neutrophils were exposed to the drug in vitro. Two other groups, however, have described inhibition of chemotaxis with methotrexate in vitro [5,18]. One of these groups used conditions similar to those of the present report, and found values

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

ANTIFOLATE 10-DEAZAAMINOPTERIN

387

FIG. 6: Inhibition of chemotaxis by methotrexate as determined by the boyden chamber technique after 110 min incubation at 37°C. Each point represents the average of 3 experiments (error bars = standard error of the mean value). (Data from Cream and Pole [5]).

for maximal inhibition and ED,, comparable to those we report here for 10-DAM [5] (Figure 6). Generally, agarose gels or the Boyden chamber technique are used for the determination of chemotaxis. In account for the seemingly inconsistent results it is possible that an inhibition of chemotaxis under certain conditions escapes the detection by the method using agarose gels [17]. Methotrexate has been reported to inhibit pagocytosis at nanomolar concentrations in a dose dependent manner [13] (Figure 7). The phenomenon was completely reversible by washing. The effect of methotrexate on superoxide generation by intact neutrophils was measured with the c y t o c h r o m e c reduction assay described above. We found methotrexate to be inhibitory, producing a maximal inhibition of 40% with a half-maximal effective dose of 40 uM (Figure 7). Like 10-DAM, methotrexate also inhibited superoxide generation in a cell-free preparation of preactivated neutrophils (shown as the open point in Figure 8).

3 88

WEBER, NAIR, AND M C CORD

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

70

0

0

1

I

I

0.5

1

1.5

2

methotrexate (micromolar)

FIG. 7: Inhibition of phagocytosis of S. aureus by neutrophils. Each point represents the average of 5 experiments (error bars = standard error of the mean value). (Data from Hyams et al. [13])

.-

50

c U 3

U

fQ 40 0)

v .5

ki n 3

rn 0

c

l z

.-c R

30

lotli'

20

O

0

,

,

50

100

,I 150

200

Methotrexate (micromolar)

FIG. 8: Inhibition of PMN superoxide generation by methotrexate. Each point represents the average of 10 individuals, except at 10 uM, where n = 4 (control value 3.72 +- 1.36 nmol 0,-/106 cells/min; error bars = standard error of the mean value). The open circle shows the result of experiments with a cell-free system (average of 5 experiments)

ANTlFOLATE 10-UEAZAAMINOPTEKIN

20

389

-

c 0 .c

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

0 3

c ._ n ._ A= .-c

-

-10

8 0

I

I

I

I

I

I

100

200

300

400

500

600

700

folic acid (micromolar)

FIG. 9 : Inhibition of PMN superoxide generation by folic acid. Each point represents the average of 10 individuals (control value 3.72 +- 1.36 nmol 0,-/106 cells/min; error bars = standard error of the mean value).

Hyams et al. [ 1 3 ] reported the inhibition by methotrexate of intracellular protein iodination by polymorphonuclear leukocytes at concentrations between 0.15 uM and 1.65 uM. This appears to be in conflict with their finding that bactericidal activity could be suppressed to a statistically significant extent (although still by less than 20%) only at the highest concentration used in that study (1.65 uM). Possibly, direct and separate measurements of free radical generation informative.

and

degranulation would

have

been

more

aureus by Comparison to Folic Acid. Phagocytosis of S . polymorphonuclear leukocytes was not impaired by 1.70 uM folic acid in an earlier study [13]. We measured the influence of folic acid 3n superoxide generation found only modest inhibition up to circa 10% (Figure 9 ) .

DISCUSSION In the cascade of neutrophil physiology: phagocytosis and bacterial killing there is a

chemotaxis, significant

390

WEBER, NAIR, AND MC CORD

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

inhibitory influence of the antifolate 10-deazaaminopterin in vitro. The effect is more than modest since an inhibition of the migration of white blood cells to the site of inflammation (e.g. a joint in rheumatoid arthritis) reduces the effects of the following processes (phagocytosis and bacterial killing) by roughly the same percentage. In combination with a 30% inhibition of the NADPH oxidase this results in an overall decrease of the damaging effects (release of lysosomal enzymes and generation of free radicals) by 60%.

The immediate inhibition of superoxide generation without preincubation suggests that at least part of the antiinflammatory effect of the drug is exerted by an interaction with the NADPH oxidase rather than by antagonistic effects on folic acid metabolism. A maximum of the inhibition of circa 30% is unusual. As breaking of the cells did not abolish the inhibition by 10-DAM, an indirect effect on the cascade of events which lead to generation of superoxide seems unlikely. Instead, it supports the hypothesis that the NADPH oxidase is a complex enzyme which can transfer electrons from NADPH to oxygen via different pathways, some of which are inhibited by 10-deazaaminopterin. With regard to the k,,, of NADPH oxidase for NADPH, which was reported to be between 20 uM and 40 uM [3], 10-DAM appears to be a powerful inhibitor of the enzyme. Some folic acid antagonists, like methotrexate [2] and 10-DAM [21], are metabolized by polyglutamylation, and thus may become pharmacologically more efficient in tumor therapy [lo]. It is not yet known, however, whether neutrophils metabolize 10deazaaminopterin. Our results show that the influence of 10-DAM on neutrophil physiology is a direct one, which seems to be unrelated to the inhibition of dihydrofolate reductase and, in case the drug is metabolized, is limited rather than improved by chemical modification inside the cells. It is also not clear if the demonstrated neutrophil inhibiting activities of the folic acid antagonists will contribute to anti-inflammatory activity in vivo, as the concentrations required for half-maximal effect exceed those currently being used in the treatment of chronic inflammatory diseases. However, it is a common observation that inhibitory drug effects on neutrophils in vitro occur at much higher doses than the comparable effects after application to humans, for e.g. the in

391

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

ANTIFOLATE 10-DEAZAAMINOPTERIN

vitro inhibition by pentoxifylline of neutrophil superoxide generation has its half-maximal effect at 500 uM [8] whereas therapeutic blood concentrations are usually under 50 uM [7]. Also, the inhibition of several neutrophil functions by non-steroidal antiinflammatory agents, like piroxicam, has half-maximally effective concentrations between 50 uM and 3 mM [l] which is well above the level that can be reached in vivo. Only an investigation into the influence of 10-DAM on neutrophils after its application to humans can lead to a decision whether the effects described in this investigation make a major contribution to the antiinflammatory activity of 10-DAM in the clinical application.

Acknowledgements: This investigation was supported by grants CA 27101 and CA 32687 (M. G. Nair) from the National Cancer Institute, USPHS/Department of Health and Human Services, and by a fellowship (G. F. Weber) from the Deutsche Forschungsgemeinschaft (Federal Republik of Germany).

'

abbreviations: HBSS Hank's balanced salt solution; PMA phorbol12-myristate-13-acetate; PMN polymorphonuclear leukocyte; 10-DAM 10-deazaaminopterin

References 1. Abramson, S.B. and Weissmann, G., The mechanisms of action of nonsteroidal antiinflammatory drugs, Arthritis & Rheumatism 32:l-9, 1989 2. Baugh, C.M., Krumdiek, C.L., and Nair, M.G., Polygammaglutamyl metabolites of methotrexate, Biochem Biophys Res Commun 52:27-34,

1973 3. Bellavite, P., The superoxide-forming enzymatic system of phagocytes, Free Rad Biol Med 4:225-261, 1988 4 . Cohen, H.J. and Chovaniec, M . E . , Superoxide generation by digitonin-stimulated guinea pig granulocytes, J Clin Invest

61: 1081-1087 , 1978

5. Cream, J.J. and Pole, D.S., The effect of methotrexate and hydroxyurea on neutrophil chemotaxis, Brit J Dermatol 102:557-563, 1980 6. DeGraw, J.I., Brown, V.H., Tagawa, H., Kisliuk, R.L., Gaumont, Y., and Sirotniak, F.M., Synthesis and antitumor activity of 10-alkyl-10-deazaminopterins. A convenient synthesis of 10-deazaminopterin, J Med Chem 25:1227, 1982

392

WEBER, NAIR, AND MC CORD

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

7. Dezube, B.J., Eder, J.P., and Pardee, A., Phase I trial of escalating pentoxifylline dose with constant dose of thiotepa, Cancer Res 50:6806-6810, 1990 8. Freyburger, G., Hammerschmidt, D.E., Coppo, P.A., and Boisseau, M.R., Leukocyte activation and rheological changes: effect of pentoxifylline. In: Pentoxifylline and leukocyte function, edited by G.L. Mandell and W.J. Novick, pp. 82-95, Somerville, N.J. 1988

9. Gilliland, B.C. and Mannik, M., Rheumatoid arthritis. In: Harrison's principles of internal medicine, edited by K.J. Isselbacher, D.A. Raymond, E. Braunwald, R.G. Petersdorf and J.D. Wilson, pp. 1872-1880, McGraw-Hill, New York et al., 1980 10. Goldman, I.D., Proceedings of the second workshop on folyl and antifolyl polyglutamates, Praeger Scientific, New York, 1985 11. Hall, C.W., Liebaers, I., Di Natale, P., and Neufeld, E.F., Enzymic diagnosis of the genetic mucopolysaccharide storage disorders. In: Methods in Enzymology. Volume L. Complex Carbohydrates. Part C, edited by V. Ginsburg, pp. 4390-456, Academic Press, New York,San FranciscotLondon,1978

12. Hohn, D.C. and Lehrer, R.I., NADPH oxidase deficiency in X-linked chronic granulomatous disease, J Clin Invest 55:707-713, 1975 13. Hyams, J.S. , Donaldson, M.H. , Metcalf , J.A. , and Root, R.K. , Inhibition of human granulocyte function by methotrexate, Cancer Res 38:650-655, 1978 14. Jandl, R.C., Andre'-Schwartz, J., Borges-DuBois, L., Kipnes, R.S., McMurrich, B.J., and Babior, BM, Termination of the respiratory burst in human neutrophils, J Clin Invest 1989 15. Jones, H.P. , Ghai, G. , Petrone, W.F. , and McCord, J.M. , Calmodulin-dependent stimulation of the NADPH oxidase of human neutrophils, Biochim Biophys Acta 714:152-156, 1982 16. McCord, J.M. and Salin, M.L., Self-directed cytotoxicity of phagocyte-generated superoxide free radical. In: Movement, Metabolism and Bactericidal Mechanisms of Phagocytes, edited by F. Rossi, P. Patriarca and D. Romeo, pp. 257-264, Piccin Medical Books, Padova, 1977 17. MacFadden, D.K., Saito, S., and Pruzanski, W., The effect of chemotherapeutic agents on chemotaxis and random migration of human leukocytes, J Clin Oncology 3:415-419, 1985 18. Melby, K. and Quie, P.G., Effects of methotrexate, ampicillin and gentamicin alone and in combination on the in vitro locomotion on human polymorphonuclear cells (PMNs), Acta Path Microbiol Immunol Scand 92, Section C:331-333, 1984 19. Michell, R.H., Karnovsky, M.J., and Karnovsky, M.L., The distribution of some granule-associated enzymes in guinea-pig polymorphonuclear leukocytes, Biochem J 116:207-216, 1970

ANTIFOLATE 10-DEAZAAMINOPTERIN

393

20. Nair, M . G . , Synthesis of 10-deazaaminopterin (10-DAAM) and 10-ethyl-lO-deazaaminopterin (10-EDAAM), J Org Chem 50:1879, 1985

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Johann Christian Senckenberg on 11/13/14 For personal use only.

21. Nair, M.G., Nanavati, N.T., Kumar, P., Gaumont, Y., R. L. , Synthesis and biological evaluation Kisliuk, 10-deazaaminopterin poly-gamma-glutamyl metabolites of lo-ethyl-deazaaminopterin, J Med Chem 31:181-185, 1988

and of and

22. O'Callaghan, J.W., Forrest, M.J., and Brooks, PM, Inhibition of neutrophil chemotaxis in methotrexate-treated rheumatoid arthritis patients, Rheumatol Int 8:41-45, 1988 23. Thompson, E. and Sarab-Al-Nakeeb, Effect of leukaemia therapy on neutrophil chemotaxis, J Clin Path 34:371-376, 1981

The antifolate 10-deazaaminopterin inhibits neutrophil chemotaxis and superoxide generation.

The effect of the dihydrofolate reductase inhibitor 10-deazaaminopterin on several neutrophil functions was tested in vitro. At 100 uM it inhibited ch...
646KB Sizes 0 Downloads 0 Views