THE JOURNAL OF INFECTIOUS DISEASES. VOL. 138, NO.4. OCTOBER 1978 © 1978 by The University of Chicago. 0022-1899178/3804-0002$00.81
Enhancement of Neutrophil Chemotaxis and Alteration of Levels of Cellular Cyclic Nudeotides by Levamisole From the Division of Clinical Immunology of the Department of Pediatrics and the Department of Pathology, University of Utah, Salt Lake City, Utah
Naney A. Hogan and Harry R. Hill
In previous studies imidazole, the parent molecule of levamisole, has been shown to enhance neutrophil chemotaxis [1]. Since imidazole has been reported to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) phosphodiesterase activity while inhibiting breakdown of guanosine 3':5'-cyclic phosphate (cyclic GMP) [2], we sought to determine if levamisole might also exert its effect through altering levels of cellular cyclic nucleotides. In addition, we have determined the effects of levamisole on the chemotactic responsiveness of polymorphonuclear leukocytes (PMNLs) from a number of patients with defective chemotaxis and recurrent infections. We report that levamisole enhanced the chemotactic response of PMNLs from patients
and controls. The compound also elevated levels of cellular cyclic GMP, especially in the presence of a chemotactic factor, and lowered the concentration of cyclic AMP.
Materials and Methods
Clinical specimens. Approximately 100 ml of heparinized blood (20 units of heparinjrnl) was drawn from healthy adult volunteers. Erythrocytes were allowed to sediment for 1 hr, after which the leukocyte-rich plasma was removed. The cells in this suspension were then used in the studies of chemotaxis and in the assays for cyclic nucleotides. Chemotaxis. Chemotaxis was studied by a modification of the procedure of Boyden [3, 4]. PMNLs from leukocyte-rich plasma were diluted in tissue culture medium 199 (Microbiological Associates, Bethesda, Md.) to a range of 5-15 X 106 PMNLs/ml of medium 199. A known quantity (0.1 ml) of the cell suspension was then diluted to 0.4 ml in medium 199 and delivered to a Millipore filter (pore size, 5 /Lm; Millipore Corp., Bedford, Mass.) utilizing a cytocentrifuge (Shandon Scientific Co., Sewickley, Pa.),
Received for publication June 13, 1977, and in revised form April 10, 1978. This study was supported in part by grants no. AI 13150, AM 18354, and AM 21140 from the U.S. Public Health Service and by a grant from the Thrasher Foundation. Dr. Hill is an investigator of the Howard Hughes Medical Institute. Please address requests for reprints to Nancy A. Hogan, Department of Pediatrics, University of Utah College of Medicine, Salt LakeCity, Utah 84132.
437
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on August 31, 2015
Levamisole, an antihelminthic agent reported to enhance nonspecifically various parameters of the immune response, was examined for its effect on chemotaxis of human neutrophils and on levels of cellular cyclic nucleotides. This agent was found, in most instances, to enhance chemotactic responses of neutrophils to a bacterial chemotactic factor derived from Escherichia coli. At similar concentrations, levamisole produced increases in levels of guanosine 3':5'-cyclic phosphate in neutrophils. In contrast, a decrease in concentrations of adenosine 3':5'-cyclic phosphate was observed when neutrophils were incubated with levamisole. Neutrophil chemotaxis, with and without the addition of levamisole, was assessed in 10 patients with recurrent infections. The illnesses of these patients included Job's syndrome, Wiskott-Aldrich syndrome, eczema with an increased level of IgE and recurrent abscesses, chronic mucocutaneous candidiasis, and diabetes mellitus. Levamisole significantly enhanced chemotaxis of polymorphonuclear leukocytes from these patients. Levamisole appears to have a profound effect on chemotactic responses of neutrophils which probably results from alterations in cellular cyclic nucleotide levels. Levamisole may prove to be useful therapeutically in certain patients with defective neutrophil chemotaxis.
438
Hanks' balanced salt solution (HBSS; Difco, Detroit, Mich.) was added, and cells were incubated with shaking for various times. Incubation was terminated by exposure of the glass tubes containing cells to the flame of a bunsen burner for 2 sec [6]. This rapid heat-killing method results in lower basal levels of cyclic nucleotides, better overall recovery (mean ± SE, 95.0% ± 2.3%), and increased sensitivity in detecting changes in response to pharmacologic agents [6]. Samples were then sonicated and filtered (filter pore size, 0.45 JLm) to remove cellular debris. The radioimmunoassay was performed with use of standards prepared in HBSS. A IOO-JLl volume of the standard solution or of the filtered, heat-inactivated cell suspension was first added to each assay tube (10- X 75-mm glass culture tubes). Acetylation of the cyclic nucleotides was accomplished as previously described [7] by addition of 3 pJ of a freshly prepared 2: I mixture of triethylamine and acetic anhydride to each assay tube. Antibody and iodinated cyclic nucleotides (Collaborative Research, Waltham, Mass.) were prepared in 50 mM sodium acetate buffer, pH 4.75, containing 20 mM CaC12 ; 100 JLl each of antibody and iodinated cyclic nucleotide was added to the assay tubes. Duplicate samples were incubated overnight at 4 C, and separation of antibody-bound cyclic nucleotide was performed by Millipore filtration [8]. Filters were then counted in a Beckman biogamma counter (Beckman Instruments, Irvine, Calif.) to a constant 4,000 cpm to eliminate the necessity of adjusting the concentration of iodinated cyclic nucleotide during radioactive decay of these compounds. Time values obtained were analyzed using a HewlettPackard model no. 10 calculator (Hewlett-Packard, Palo Alto, Calif.) equipped with a radioimmunoassay program. Results
Levamisole markedly enhanced chemotactic responses of PMNLs to the E. coli bacterial factor for the majority of control subjects studied. As shown in figure I, increases in chemotaxis ranged from 30% to 120% with 2.5 X 10-4 M levamisole and from 70% to 190% with 5 X 10-4 M levamisole. PMNLs from three normal subjects failed to show an increase in chemotactic re-
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on August 31, 2015
The filters were then placed in a modified Boyden chamber (Neuroprobe Corp., Bethesda, Md.), and a chemotactic stimulus was added to the attractant side. After incubation for 3 hr at 37 C, the filters were removed, fixed in methanol, stained with hematoxylin, dehydrated with alcohol, and cleared with xylene. The number of cells that had migrated completely through the filter in 10 random fields was determined microscopically using a X 10 ocular, x45 objective, and 5- X 5-mm photographic reticule. A chemotactic index was then calculated by dividing the number of PMNLs that had migrated completely through the filter within the reticule in 10 random fields by the total number of PMNLs (X 106 ) in the O.4-ml cell suspension delivered to the starting side of the filter. Chemotactic factor. A bacterial chemotactic factor was prepared from a culture filtrate of Escherichia coli grown in medium 199 for 24 hr at 37 C [5]. After passage through a micropore filter (pore size, 0.22 JLm), this bacterial factor was frozen at -70 C in l-ml aliquots. The bacterial factor was diluted to 50 JLl/ml of medium 199, and -2 ml of this solution was added to the hottom or attractant side of the chemotactic chamber. Medium 199 alone or medium 199 plus the test agent was added to the top side of the filter. In experiments comparing spontaneous random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis), various concentrations of the bacterial chemotactic factor were added to both sides of the filter, with and without the addition of levamisole, to achieve the desired concentration gradients. Cyclic nucleotide assays. Purified preparations of PMNLs (98% PMNLs, 2% mononuclear cells) were obtained by density gradient centrifugation of the leukocyte-rich plasma on FicollHypaque (Pharmacia Fine Chemicals, Piscataway, N.].) as previously described [6, 7]. PMNLs were washed once and adjusted to a concentration of 5 X 106/m!. All incubations were performed using 1.0 ml of cell suspension in 15- X IOO-mm glass culture tubes. The experiments were performed in triplicate for each condition, with duplicate assays in each experiment. The cells were preincubated for 20 min at 37 C in a water bath (New Brunswick Scientific, New Brunswick, N.J.). A IO-JLl aliquot of test agent or
Hogan and Hill
439
Levamisole and PMNL Chemotaxis
Table 1. Effect of 5 X 10-4 M levamisole on spontaneous random migration, chemokinesis, and chemotaxis of polymorphonuclear leukocytes from normal humans in response to a bacterial factor prepared from Escherichia coli.
280
CJl
x ~
200
EC
~
Bacterial factor (%) below filter
W
:I:
U
Bacterial factor (%) above filter
Z
JH
~ 120
Enhancement of Neutrophil Chemotaxis and Alteration of Levels of Cellular Cyclic Nudeotides by Levamisole From the Division of Clinical Immunology of the Department of Pediatrics and the Department of Pathology, University of Utah, Salt Lake City, Utah
Naney A. Hogan and Harry R. Hill
In previous studies imidazole, the parent molecule of levamisole, has been shown to enhance neutrophil chemotaxis [1]. Since imidazole has been reported to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) phosphodiesterase activity while inhibiting breakdown of guanosine 3':5'-cyclic phosphate (cyclic GMP) [2], we sought to determine if levamisole might also exert its effect through altering levels of cellular cyclic nucleotides. In addition, we have determined the effects of levamisole on the chemotactic responsiveness of polymorphonuclear leukocytes (PMNLs) from a number of patients with defective chemotaxis and recurrent infections. We report that levamisole enhanced the chemotactic response of PMNLs from patients
and controls. The compound also elevated levels of cellular cyclic GMP, especially in the presence of a chemotactic factor, and lowered the concentration of cyclic AMP.
Materials and Methods
Clinical specimens. Approximately 100 ml of heparinized blood (20 units of heparinjrnl) was drawn from healthy adult volunteers. Erythrocytes were allowed to sediment for 1 hr, after which the leukocyte-rich plasma was removed. The cells in this suspension were then used in the studies of chemotaxis and in the assays for cyclic nucleotides. Chemotaxis. Chemotaxis was studied by a modification of the procedure of Boyden [3, 4]. PMNLs from leukocyte-rich plasma were diluted in tissue culture medium 199 (Microbiological Associates, Bethesda, Md.) to a range of 5-15 X 106 PMNLs/ml of medium 199. A known quantity (0.1 ml) of the cell suspension was then diluted to 0.4 ml in medium 199 and delivered to a Millipore filter (pore size, 5 /Lm; Millipore Corp., Bedford, Mass.) utilizing a cytocentrifuge (Shandon Scientific Co., Sewickley, Pa.),
Received for publication June 13, 1977, and in revised form April 10, 1978. This study was supported in part by grants no. AI 13150, AM 18354, and AM 21140 from the U.S. Public Health Service and by a grant from the Thrasher Foundation. Dr. Hill is an investigator of the Howard Hughes Medical Institute. Please address requests for reprints to Nancy A. Hogan, Department of Pediatrics, University of Utah College of Medicine, Salt LakeCity, Utah 84132.
437
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on August 31, 2015
Levamisole, an antihelminthic agent reported to enhance nonspecifically various parameters of the immune response, was examined for its effect on chemotaxis of human neutrophils and on levels of cellular cyclic nucleotides. This agent was found, in most instances, to enhance chemotactic responses of neutrophils to a bacterial chemotactic factor derived from Escherichia coli. At similar concentrations, levamisole produced increases in levels of guanosine 3':5'-cyclic phosphate in neutrophils. In contrast, a decrease in concentrations of adenosine 3':5'-cyclic phosphate was observed when neutrophils were incubated with levamisole. Neutrophil chemotaxis, with and without the addition of levamisole, was assessed in 10 patients with recurrent infections. The illnesses of these patients included Job's syndrome, Wiskott-Aldrich syndrome, eczema with an increased level of IgE and recurrent abscesses, chronic mucocutaneous candidiasis, and diabetes mellitus. Levamisole significantly enhanced chemotaxis of polymorphonuclear leukocytes from these patients. Levamisole appears to have a profound effect on chemotactic responses of neutrophils which probably results from alterations in cellular cyclic nucleotide levels. Levamisole may prove to be useful therapeutically in certain patients with defective neutrophil chemotaxis.
438
Hanks' balanced salt solution (HBSS; Difco, Detroit, Mich.) was added, and cells were incubated with shaking for various times. Incubation was terminated by exposure of the glass tubes containing cells to the flame of a bunsen burner for 2 sec [6]. This rapid heat-killing method results in lower basal levels of cyclic nucleotides, better overall recovery (mean ± SE, 95.0% ± 2.3%), and increased sensitivity in detecting changes in response to pharmacologic agents [6]. Samples were then sonicated and filtered (filter pore size, 0.45 JLm) to remove cellular debris. The radioimmunoassay was performed with use of standards prepared in HBSS. A IOO-JLl volume of the standard solution or of the filtered, heat-inactivated cell suspension was first added to each assay tube (10- X 75-mm glass culture tubes). Acetylation of the cyclic nucleotides was accomplished as previously described [7] by addition of 3 pJ of a freshly prepared 2: I mixture of triethylamine and acetic anhydride to each assay tube. Antibody and iodinated cyclic nucleotides (Collaborative Research, Waltham, Mass.) were prepared in 50 mM sodium acetate buffer, pH 4.75, containing 20 mM CaC12 ; 100 JLl each of antibody and iodinated cyclic nucleotide was added to the assay tubes. Duplicate samples were incubated overnight at 4 C, and separation of antibody-bound cyclic nucleotide was performed by Millipore filtration [8]. Filters were then counted in a Beckman biogamma counter (Beckman Instruments, Irvine, Calif.) to a constant 4,000 cpm to eliminate the necessity of adjusting the concentration of iodinated cyclic nucleotide during radioactive decay of these compounds. Time values obtained were analyzed using a HewlettPackard model no. 10 calculator (Hewlett-Packard, Palo Alto, Calif.) equipped with a radioimmunoassay program. Results
Levamisole markedly enhanced chemotactic responses of PMNLs to the E. coli bacterial factor for the majority of control subjects studied. As shown in figure I, increases in chemotaxis ranged from 30% to 120% with 2.5 X 10-4 M levamisole and from 70% to 190% with 5 X 10-4 M levamisole. PMNLs from three normal subjects failed to show an increase in chemotactic re-
Downloaded from http://jid.oxfordjournals.org/ at Cambridge University on August 31, 2015
The filters were then placed in a modified Boyden chamber (Neuroprobe Corp., Bethesda, Md.), and a chemotactic stimulus was added to the attractant side. After incubation for 3 hr at 37 C, the filters were removed, fixed in methanol, stained with hematoxylin, dehydrated with alcohol, and cleared with xylene. The number of cells that had migrated completely through the filter in 10 random fields was determined microscopically using a X 10 ocular, x45 objective, and 5- X 5-mm photographic reticule. A chemotactic index was then calculated by dividing the number of PMNLs that had migrated completely through the filter within the reticule in 10 random fields by the total number of PMNLs (X 106 ) in the O.4-ml cell suspension delivered to the starting side of the filter. Chemotactic factor. A bacterial chemotactic factor was prepared from a culture filtrate of Escherichia coli grown in medium 199 for 24 hr at 37 C [5]. After passage through a micropore filter (pore size, 0.22 JLm), this bacterial factor was frozen at -70 C in l-ml aliquots. The bacterial factor was diluted to 50 JLl/ml of medium 199, and -2 ml of this solution was added to the hottom or attractant side of the chemotactic chamber. Medium 199 alone or medium 199 plus the test agent was added to the top side of the filter. In experiments comparing spontaneous random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis), various concentrations of the bacterial chemotactic factor were added to both sides of the filter, with and without the addition of levamisole, to achieve the desired concentration gradients. Cyclic nucleotide assays. Purified preparations of PMNLs (98% PMNLs, 2% mononuclear cells) were obtained by density gradient centrifugation of the leukocyte-rich plasma on FicollHypaque (Pharmacia Fine Chemicals, Piscataway, N.].) as previously described [6, 7]. PMNLs were washed once and adjusted to a concentration of 5 X 106/m!. All incubations were performed using 1.0 ml of cell suspension in 15- X IOO-mm glass culture tubes. The experiments were performed in triplicate for each condition, with duplicate assays in each experiment. The cells were preincubated for 20 min at 37 C in a water bath (New Brunswick Scientific, New Brunswick, N.J.). A IO-JLl aliquot of test agent or
Hogan and Hill
439
Levamisole and PMNL Chemotaxis
Table 1. Effect of 5 X 10-4 M levamisole on spontaneous random migration, chemokinesis, and chemotaxis of polymorphonuclear leukocytes from normal humans in response to a bacterial factor prepared from Escherichia coli.
280
CJl
x ~
200
EC
~
Bacterial factor (%) below filter
W
:I:
U
Bacterial factor (%) above filter
Z
JH
~ 120
