258
Agents and Actions vol. 8/3 (1978) Birkh~iuser Verlag, Basel
The Neutrophil Adherence Assay as a Method for Detecting Unique Anti-Inflammatory Agents by VERA J. STECHER 1 and GEORGE L. CHINEA Research Department, Pharmaceuticals Division, CIBA-Geigy Corporation, Ardsley, New York 10502, USA
Abstract Neutrophil adhesiveness is an important eomponent of the pathophyslology of the inflammatory process. Modulation of this funetlon eould result in a reduetion in the degree of inflammation and connective tissue damage. A simple technique using filtration of whole blood through nylon fiber columns has been developed to study the effect of anti-inflammatory agents on the adherence of rat peripheral blood neutrophils. Gold and chloroquiue, in addition to many standard anti-inflammatory drugs, have been shown to cause inhibition of neutrophil adherence. Measurement of this parameter of cell behavior has the potential of detecting anti-inflammatory agents not normally effective in the standard animal models.
During the inflammatory response, circulating neutrophils adhere to the venular endothelium and migrate into the tissue. There is little doubt that the accumulation o f neutrophils is an important component in the pathogenesis of rheumatoid arthritis. ZVAIFLER [1], for example, has pointed out that in the rheumatoid joint the accumulation of 500 million leukocytes per 10 ml effusion is a characteristic of this inflammatory response. GRANT [2] has extensively reviewed the adherence and emigration of white blood cells in inflammation. Modulation of neutrophil adherence therefore might be a realistic therapeutic goal that could result in a reduction in the degree of inflammation. There is a need for an assay that will detect not only 'classic' nonsteroidal anti-inflammatory drugs, but gold and chloroquine-like compounds as well. This paper describes such an assay procedure, which combines the relevancy o f 5 days of 1Professor, Union Graduate School and Adj. Assoc. Professor, New York Medical College.
in vivo dosing of animals with the precision o f in vitro analysis of the test agent/active metabolite effect on leukocyte adherence. It has been shown by MACGREGOR et al. [3] that peripheral blood granulocytes from h u m a n volunteers who received prednisone or aspirin had a reduced ability to adhere to nylon fiber columns packed in Pasteur pipettes. The technique of MacGregor requires Pasteur pipette tips to' be heated and drawn out to a critical length and tip opening. Likewise, a method reported by GARV~ [4] for studying adhesiveness of h u m a n leukocytes required an elaborate column attached to a motor-driven syringe. Described in this report is a rapid, simple and reproducible method for determining the adhesiveness of peripheral blood neutrophils from rats treated with antiinflammatory agents. Materials and methods
Figure 1 illustrates the adherence column used in these studies. It consists of a 1 ml disposable tuberculin syringe (Becton, Dickinson, Rutherford, N.J.) packed to the 0.25 ml mark with 80 mg scrubbed nylon fiber (FT-242 FenwaU Laboratories, Morton Grove, IlL) and fitted with a stopcock (B-D MS01) and 25-gauge ~ inch disposable needle. Heparinized blood (preservative-free heparin) was taken from the abdominal aorta of each treated rat and was kept cold prior to being placed on the adherence column. Five animals were used in each treatment group. The packed columns were pre-ineubated at 37~ for 10 minutes. After this periOd of warming, 1 ml of blood was placed on the columns using a tuberculin syringe fitted with a 14 G, 4 inch needle (B-D Special Needle 01 0021) and incubated for 5 minutes at 37~ The stopcocks were opened and the blood allowed to filter through the scrubbed nylon fiber by gravitational force for an additional 10 minutes. A control aliquot of unfiltered blood was incubated in tubes for the same length of time. Following the incubation time, and prior to
The Neutrophil Adherence Assay as a Method for DetectingUnique Anti-InflammatoryAgents Adherence Column
1.0cc Tuberculin Syringe
Nylon Fiber
- - t - W a y
Stopcock (closed)
25g 5/~Needle
-
-
,Collecting Tube
Figure 1 Column used to study leukocyte adherence consists of a disposable 1 cc tuberculin syringe packed to the 0.25 ec mark with 80 mg of scrubbed nylon fiber and closed with a one-way stopcock.
counting, the blood samples were kept on ice. Total blood leukocyte counts were determined with the Coulter Counter| Model ZBI, and differential counts (200 cells/slide, 2 slides/sample) were taken on the filtered blood and unfiltered control blood. The percent of neutrophils was multiplied by the total white blood cell counts to determine the total number of neutrophils/mrn3. The values obtained for blood filtered through the nylon fiber column were subtracted from those values of the unfiltered blood for each animal. The percent of neutrophils retained on the adherence columns in the treated group was compared with that of the untreated control group and percent inhibition of adherence was calculated. With the exception of gold, all the drugs listed in Figures 2 and 3 were given orally for 5 days; the final dose being given 1 hour prior to the test. The drugs were homogenized in an aqueous vehicle consisting of 3% cornstarch, 5% polyethylene glycol 400 and 2 drops of Tween 80. The untreated control groups received vehicle alone. Charles River (COBS-CD) male rats, weighing 225 to 235 g, were used exclusively in this study. Gold sodium thiomalate, Myochrysine| was administered by intra-muscular injection (25 mg/kg).
259
Results Figure 2 illustrates the percent inhibition of neutrophil adherence produced by anti-inflammatory agents in comparison with nontreated control animals. The number of experiments performed is given in parentheses above each column, and the statistical significance given by the p value was calculated using the Student's 7test. While the standard anti-inflammatory drugs are picked up by this assay, Figure 2 shows quite clearly that in addition both gold (i.m. 25 mg/kg) and chloroquine (p.o. 25 mg/kg) are detected. The adherence assay, performed as described, has provided a method for detecting an activity of various types of anti-inflammatory compounds. In most cases, the dosages used in this assay (Fig. 2) are within the known effective antiinflammatory activity range in rats. Furthermore, no effect of the drugs on total peripheral blood leukocyte count was noted over the 5-day dosing period. Table 1 illustrates that there is a doseresponse relationship in the effect of aspirin on neutrophil adherence. The higher the dose, the lower the number of neutrophils which adhered to the nylon fiber. At 25 mg/kg p.o. the percent of neutrophils adhering approached control values. The dose-response pattern seen with aspirin appears to be typical of that obtained with other anti-inflammatory compounds. The effects of additional types of antiinflammatory compounds on neutrophil adherence are shown in Figure 3. All the drugs were studied in at least two separate experiments. Surprisingly, there was a dramatic difference in the percent inhibition of neutrophil adherence caused by D-L-penicillamine as compared with the effect of D-penicillamine. This data might provide an explanation for the finding of WATNICK [5] that D-L-penicillamine (100 and 150 mg/kg p.o.) decreased the chronic inflammatory response in the non-injected paw of adjuvant arthritic rats. On the other hand, orally administered o-penicillamine (100 and 150 mg/kg) had no significant effects in this model. It may therefore be possible to use the neutrophil adherence assay to distinguish new compounds with similar anti-inflammatory profiles. It is also interesting to note that two known anti-inflammatory drugs, flumefinine and prodolic acid, are however inactive in inhibiting neutrophil adherence. These drugs may be acting on parameters which appear later in the inflammatory response.
260
The Neutrophil Adherence Assay as a Method for DetectingUnique Anti-InflammatoryAgents
Percent I nhibition of Neutrophil A d h e r e n c e % 100
-~
(N) = Number of experiments
(2)
(2) p < = 0.01
0.001
(2)
(2) 0.001
0.001
C4)
(1)
0,01
0.001
(3) o.ool
(1)
0,001
(2) N.S.
~
q,
/o
o/
,o,
oo
Dose mg/kg Figure 2
Percent inhibition of neutrophil adherence produced by antiinflammatoryagents as compared to non-treated control group. Number of experiements performed appears in parentheses above each column. Gold was giveni.m. (25 mg/kg) and all other drugs were administered p.o. at the doses indicated. cumulation in areas of inflammation should therefore help to reduce the severity of the disease. Inflammatory disease states are known to Treatment Dose % of neutrophils Significance (mg/kg p.o.) adhering (P) cause an enhancement of neutrophil adherence [6]. MACGREGOR [7] has reported that human 3% Cornstarch 1 ml/100 gbw 77.9 + 2.7 rheumatoid arthritic patients have a significantly enhanced level of granulocyte adherence in Aspirin 100 57.1 + 3.9
Agents and Actions vol. 8/3 (1978) Birkh~iuser Verlag, Basel
The Neutrophil Adherence Assay as a Method for Detecting Unique Anti-Inflammatory Agents by VERA J. STECHER 1 and GEORGE L. CHINEA Research Department, Pharmaceuticals Division, CIBA-Geigy Corporation, Ardsley, New York 10502, USA
Abstract Neutrophil adhesiveness is an important eomponent of the pathophyslology of the inflammatory process. Modulation of this funetlon eould result in a reduetion in the degree of inflammation and connective tissue damage. A simple technique using filtration of whole blood through nylon fiber columns has been developed to study the effect of anti-inflammatory agents on the adherence of rat peripheral blood neutrophils. Gold and chloroquiue, in addition to many standard anti-inflammatory drugs, have been shown to cause inhibition of neutrophil adherence. Measurement of this parameter of cell behavior has the potential of detecting anti-inflammatory agents not normally effective in the standard animal models.
During the inflammatory response, circulating neutrophils adhere to the venular endothelium and migrate into the tissue. There is little doubt that the accumulation o f neutrophils is an important component in the pathogenesis of rheumatoid arthritis. ZVAIFLER [1], for example, has pointed out that in the rheumatoid joint the accumulation of 500 million leukocytes per 10 ml effusion is a characteristic of this inflammatory response. GRANT [2] has extensively reviewed the adherence and emigration of white blood cells in inflammation. Modulation of neutrophil adherence therefore might be a realistic therapeutic goal that could result in a reduction in the degree of inflammation. There is a need for an assay that will detect not only 'classic' nonsteroidal anti-inflammatory drugs, but gold and chloroquine-like compounds as well. This paper describes such an assay procedure, which combines the relevancy o f 5 days of 1Professor, Union Graduate School and Adj. Assoc. Professor, New York Medical College.
in vivo dosing of animals with the precision o f in vitro analysis of the test agent/active metabolite effect on leukocyte adherence. It has been shown by MACGREGOR et al. [3] that peripheral blood granulocytes from h u m a n volunteers who received prednisone or aspirin had a reduced ability to adhere to nylon fiber columns packed in Pasteur pipettes. The technique of MacGregor requires Pasteur pipette tips to' be heated and drawn out to a critical length and tip opening. Likewise, a method reported by GARV~ [4] for studying adhesiveness of h u m a n leukocytes required an elaborate column attached to a motor-driven syringe. Described in this report is a rapid, simple and reproducible method for determining the adhesiveness of peripheral blood neutrophils from rats treated with antiinflammatory agents. Materials and methods
Figure 1 illustrates the adherence column used in these studies. It consists of a 1 ml disposable tuberculin syringe (Becton, Dickinson, Rutherford, N.J.) packed to the 0.25 ml mark with 80 mg scrubbed nylon fiber (FT-242 FenwaU Laboratories, Morton Grove, IlL) and fitted with a stopcock (B-D MS01) and 25-gauge ~ inch disposable needle. Heparinized blood (preservative-free heparin) was taken from the abdominal aorta of each treated rat and was kept cold prior to being placed on the adherence column. Five animals were used in each treatment group. The packed columns were pre-ineubated at 37~ for 10 minutes. After this periOd of warming, 1 ml of blood was placed on the columns using a tuberculin syringe fitted with a 14 G, 4 inch needle (B-D Special Needle 01 0021) and incubated for 5 minutes at 37~ The stopcocks were opened and the blood allowed to filter through the scrubbed nylon fiber by gravitational force for an additional 10 minutes. A control aliquot of unfiltered blood was incubated in tubes for the same length of time. Following the incubation time, and prior to
The Neutrophil Adherence Assay as a Method for DetectingUnique Anti-InflammatoryAgents Adherence Column
1.0cc Tuberculin Syringe
Nylon Fiber
- - t - W a y
Stopcock (closed)
25g 5/~Needle
-
-
,Collecting Tube
Figure 1 Column used to study leukocyte adherence consists of a disposable 1 cc tuberculin syringe packed to the 0.25 ec mark with 80 mg of scrubbed nylon fiber and closed with a one-way stopcock.
counting, the blood samples were kept on ice. Total blood leukocyte counts were determined with the Coulter Counter| Model ZBI, and differential counts (200 cells/slide, 2 slides/sample) were taken on the filtered blood and unfiltered control blood. The percent of neutrophils was multiplied by the total white blood cell counts to determine the total number of neutrophils/mrn3. The values obtained for blood filtered through the nylon fiber column were subtracted from those values of the unfiltered blood for each animal. The percent of neutrophils retained on the adherence columns in the treated group was compared with that of the untreated control group and percent inhibition of adherence was calculated. With the exception of gold, all the drugs listed in Figures 2 and 3 were given orally for 5 days; the final dose being given 1 hour prior to the test. The drugs were homogenized in an aqueous vehicle consisting of 3% cornstarch, 5% polyethylene glycol 400 and 2 drops of Tween 80. The untreated control groups received vehicle alone. Charles River (COBS-CD) male rats, weighing 225 to 235 g, were used exclusively in this study. Gold sodium thiomalate, Myochrysine| was administered by intra-muscular injection (25 mg/kg).
259
Results Figure 2 illustrates the percent inhibition of neutrophil adherence produced by anti-inflammatory agents in comparison with nontreated control animals. The number of experiments performed is given in parentheses above each column, and the statistical significance given by the p value was calculated using the Student's 7test. While the standard anti-inflammatory drugs are picked up by this assay, Figure 2 shows quite clearly that in addition both gold (i.m. 25 mg/kg) and chloroquine (p.o. 25 mg/kg) are detected. The adherence assay, performed as described, has provided a method for detecting an activity of various types of anti-inflammatory compounds. In most cases, the dosages used in this assay (Fig. 2) are within the known effective antiinflammatory activity range in rats. Furthermore, no effect of the drugs on total peripheral blood leukocyte count was noted over the 5-day dosing period. Table 1 illustrates that there is a doseresponse relationship in the effect of aspirin on neutrophil adherence. The higher the dose, the lower the number of neutrophils which adhered to the nylon fiber. At 25 mg/kg p.o. the percent of neutrophils adhering approached control values. The dose-response pattern seen with aspirin appears to be typical of that obtained with other anti-inflammatory compounds. The effects of additional types of antiinflammatory compounds on neutrophil adherence are shown in Figure 3. All the drugs were studied in at least two separate experiments. Surprisingly, there was a dramatic difference in the percent inhibition of neutrophil adherence caused by D-L-penicillamine as compared with the effect of D-penicillamine. This data might provide an explanation for the finding of WATNICK [5] that D-L-penicillamine (100 and 150 mg/kg p.o.) decreased the chronic inflammatory response in the non-injected paw of adjuvant arthritic rats. On the other hand, orally administered o-penicillamine (100 and 150 mg/kg) had no significant effects in this model. It may therefore be possible to use the neutrophil adherence assay to distinguish new compounds with similar anti-inflammatory profiles. It is also interesting to note that two known anti-inflammatory drugs, flumefinine and prodolic acid, are however inactive in inhibiting neutrophil adherence. These drugs may be acting on parameters which appear later in the inflammatory response.
260
The Neutrophil Adherence Assay as a Method for DetectingUnique Anti-InflammatoryAgents
Percent I nhibition of Neutrophil A d h e r e n c e % 100
-~
(N) = Number of experiments
(2)
(2) p < = 0.01
0.001
(2)
(2) 0.001
0.001
C4)
(1)
0,01
0.001
(3) o.ool
(1)
0,001
(2) N.S.
~
q,
/o
o/
,o,
oo
Dose mg/kg Figure 2
Percent inhibition of neutrophil adherence produced by antiinflammatoryagents as compared to non-treated control group. Number of experiements performed appears in parentheses above each column. Gold was giveni.m. (25 mg/kg) and all other drugs were administered p.o. at the doses indicated. cumulation in areas of inflammation should therefore help to reduce the severity of the disease. Inflammatory disease states are known to Treatment Dose % of neutrophils Significance (mg/kg p.o.) adhering (P) cause an enhancement of neutrophil adherence [6]. MACGREGOR [7] has reported that human 3% Cornstarch 1 ml/100 gbw 77.9 + 2.7 rheumatoid arthritic patients have a significantly enhanced level of granulocyte adherence in Aspirin 100 57.1 + 3.9
