88
QUANTITATION OF THE INFLAMMATORY ACCUMULATION OF MONONUCLEAR PHAGOCYTES IN VIVO
George J. Cianciolo Ralph Snyderman
INTRODUCTION
Mononuclear phagocytes serve important functions in the recognition and effector limbs of immune responses as well as in inflammatory reactions and wound healing. The mechanisms by which these motile r-ells accumulate locally is still poorly
understood. Although macrophages have been shown to be capable of exhibiting chemotactic migration in vitro, there is as yet no practical way to demonstrate the migration of macrophages along a chemoattractant gradient in vivo. It is, however, possible to measure the kinetics and magnitude of the accumula- tion of macrophages at inflammatory sites in vivo. Several different methods have been employed for making such measure- ments. All of them are based on the introduction of some in-
flammatory stimulant into a well-defined tissue space and quan- tifying the macrophages that subsequently accumulate in that space. We shall describe several methods that we have success-
METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.
MONONUCLEAR PHAGOCYTES 9 5 9 All rights of reproduction in any form reserved.
ISBN 0-12-044220-5
fully utilized either to quantitate macrophage accumulation in vivo or to demonstrate the production of chemotactic ac- tivity at sites of inflammation.
II. REAGENTS
1. Inflammatory Stimulants
Phytohemagglutinin(PHA) (Burroughs-Wellcome Co., Research Triangle Park, North Carolina) 35 yg in 2.0 ml sterile isotonic saline (for mice)
Concanavalin A(con A) (3 x crystallized; Miles-Yeda Ltd., Israel) 50 pg in 2.0 m l sterile isotonic saline (for mice)
Sodium metaperiodate (NalC^) (Fisher Scientific Co., Fair Lawn, New Jersey) 1.07 m g in 1.0 ml sterile isotonic saline
(for mice)
Brewer's thioglycollate broth (Difco Laboratories, Detroit, Michigan) 4% (w/v) in deionized water, sterilized by autoclav- ing
Shellfish glycogen Type II (Sigma Chemical Co., St. Louis, Missouri) 0.5% (w/v) in sterile isotonic saline
Sodium caseinate (Difco Laboratories, Detroit, Michigan) 1% (w/v) in isotonic saline, sterilized by autoclaving
2. Media
Gey1s balanced salt solution containing 2% bovine serum al- bumin and sodium bicarbonate (Flow Laboratories, McLean, Vir- ginia) , 10 ταΜ HEPES, pH 7.0
RPMI 1640 (GIBCO, Grand Island, New Y o r k ) , 10 m M HEPES, pH 7.0
Phosphate buffered (0.01 M) saline, pH 7.0
3. Preservative-free Heparin (UpJohn Co., Kalamazoo, Michigan) added 10 p/ml for peritoneal lavage 4. Apparatus for Injections
Mouse: 5- or 10-ml syringes; 25-gauge needles Guinea pig: 30-ml syringes; 19-gauge needles 5. Materials for Peritoneal Washouts
Mouse: 70% Ethyl alcohol; (2) tooth-edged forceps; 10 ml syringes; 19-gauge needles; 30-ml polypropylene tubes
Guinea pig: Hair clipper or razor; 70% ethyl alcohol;
XI. MONONUCLEAR PHAGOCYTES IN VIVO 961 scalpel blade and surgical scissors; (2) surgical forceps;
(2) ring stands; (2) rubber spatulas; 30-ml syringes; 19-gauge needles; 50-ml polypropylene tubes
III. PROCEDURES
The kinetics and magnitude of leukocyte accumulation in vivo can be readily studied by the introduction of inflammatory
agents into the peritoneal cavity. At various times after in- troduction of the inflammatory agent, tissue culture medium is injected, the cavities vigorously lavaged, and the contents are aspirated using a syringe. Total and differential leukocyte cell counts can be determined for the peritoneal washings and the recovered cells or their supernatant fluids can be used for in vitro assays.
1. Mouse Peritoneal Macrophages
Mice are injected intraperitoneally with 2.0 ml of PHA, con A, thipglycollate broth or 1.0 ml of sodium periodate, or other inflammatory stimulants and the cells harvested after the appropriate period of time (Table I). Injections are best ac- complished using a 25-gauge or smaller needle since this mini- mizes the trauma of injection. The accumulated cells are col- lected as follows: The mouse is killed by CO2 asphyxiation or decapitation, the abdominal area is wetted with 70% ethyl alco- hol, and the skin elevated from the abdominal wall then re- tracted using two tooth-edged forceps to expose the abdominal muscle wall. Eleven ml (less if the mice are extremely small, e.g., <15 gm) of heparinized medium is vigorously injected into the lower midline using a 19-gauge needle and the fluid (^10 ml) is then carefully withdrawn using the same syringe and needle
(bevel down). The injection and withdrawal procedures are re- peated, using the same puncture site if possible. Withdrawal of the injected fluid is facilitated by tenting the abdominal wall with the needle (bevel down) as this helps to prevent blockage by viscera or omentum. The second washout will usually yield 11 ml back and the two washouts are pooled in a 30-ml polypropylene centrifuge tube. Cell counts and differential leukocyte counts can be done directly on this pooled material.
The length of time between injection and cell harvesting will depend on the purpose of the assay and, if macrophages are re- quired, the percentage of contaminating polymorphonuclear leu- kocytes (PMN) that is acceptable. Typical results obtained in our laboratory for the various stimuli are listed in Table I.
Increasing the length of time between injection and harvesting
TABLE I. Intraperitoneal Accumulation of Cells to Various Inflammatory Stimuli in Mice
Inflammatory stimulus3· None PHA con A NaI04
Thioglycollate
Hours post- inj ection"
— 48 48 72 48
Total cells0 (xlO6)
3.6 12.9 12.5 11.8 12.9
Total M0C (*10e)
1.8 8.4 8.3 6.6 11.1
Total PMNC (xlO6)
<0.05 0.6 1.5 1.3 1.0
aFive mice per group were either uninjected or injected with 2 ml of PHA (35 ]ig) , con A (50 \ig) , thiogly collate (4%) , or with 1 ml of sodium periodate (5 mM) .
"At indicated time after injection, the peritoneal cavities were lavaged twice with 11 ml of heparinized medium and total and differential cell counts performed.
cMean value for each group of five mice.
can decrease the percentage of PMN to <1% but will also de- crease total number of macrophages obtained. If the cells are to be used for in vitro assays, they are washed once (400 g, 10 min, 4°C) in heparin-free medium and resuspended. If the supernatants are to be tested for biological activity, the washout volumes can be reduced to a more suitable volume. In quantitating the effects of various agents on the peritoneal inflammatory response, experimental groups should contain a minimum of five animals.
2. Guinea Pig Peritoneal Macrophages
Guinea pigs (400 - 500 gm) are injected ip with 25 - 30 ml of glycogen, sodium caseinate, or any appropriate inflammatory stimulant. At various times after injection, depending on the level of PMN contamination determined acceptable, the cells are harvested. A specific immunologically mediated reaction can be generated by first sensitizing the animals with a specific an- tigen such as Bacillus Calmette-Guerin or horseradish peroxi- dase (1). Intraperitoneal injection of the sensitized animals with the sensitizing antigen will then result in an inflamma- tory exudate that can be harvested at appropriate times.
In order to harvest the cells (2) the animal is killed, the abdomen is shaved, washed with 70% ethyl alcohol, and an ^10-cm midline incision made. The abdominal walls are retracted using hemostats attached to ring stands. Fifty ml of heparinized medium is vigorously injected and the abdominal contents care- fully but rigorously agitated. Care must be taken to avoid rup-
XI. MONONUCLEAR PHAGOCYTES IN VIVO 963 turing any blood vessels, the liver, or the spleen. This agi- tation is facilitated by the use of small rubber spatulas such as used in the kitchen. The fluid is withdrawn from the left lower quadrant using a syringe and needle and a spatula to keep the viscera away from the needle. The injection and withdrawal are repeated several times and the washes pooled in 50-ml poly- propylene centrifuge tubes. The cells are collected by centri- fugation at 400 g for 10 min at 4°C, resuspended in heparin-free medium, and a cell count and differential performed. The volume of medium used for the washout can be reduced appropriately if the supernatant is to be tested for biological activity and the cells can be washed in heparin-free medium and used for in vitro assays. If serial samples are needed from a single animal, a silastic plastic catheter can be surgically implanted into the peritoneal cavity (1, 3). This method, while tedious to set up, permits study of the kinetics of the generation of mediators of inflammation in vivo and can be extremely valuable for certain studies.
3. Potential Pitfalls
Care must be taken to ensure that intraperitoneal injections are not given subcutaneously, or into the bowel or other organs.
Bowel perforation during injection can cause bacterial peritoni- tis.
For this reason the injected materials should be kept ster- ile when possible. When washing out the peritoneal cavities of both mice and guinea pigs, care must be taken to avoid lacerat- ing major organs or blood vessels since the accumulation of peripheral blood may interfere with the results. Even minor un- seen perforations of the intestines during the washout procedure can result in bacterial contamination, which often causes dra- matic changes in the in vitro responsiveness of the cells; the cell suspensions should be carefully examined for such contami- nation before in vitro use.
ANALYSIS OF DATA
The injection of inflammatory stimuli into the peritoneal cavities of mice or guinea pigs results in an inflammatory re- sponse, the kinetics of which can easily be studied and have been previously described (4). Table II illustrates the num- bers of macrophages or polymorphonuclear leukocytes found in the peritoneal cavities of mice injected 6, 24, 48, or 72 hr earlier with PHA. As can be seen, the PMN response is extreme- ly rapid, reaching a peak within the first 6 hr and then rapidly
TABLE II. Kinetics of Intraperitoneal Cell Accumulation in Mice after ΡΗΆ Injection3
Time after injection"
(hr) 0 6 24 48 72
Total cells0 (xlO6)
3.8 10.1 11.6 10.5 11.3
Total M0C (*106)
2.3 0.8 5.3 7.0 7.0
Total
PMNC
(xlO6) 0.01 8.9 2.5 0.6 0.5
aFive mice per group received either no injection or 2 ml of sterile saline containing 35 \\g of PHA.
"At the indicated time after inject ion, the peritoneal cavi- ties were lavaged twice with 11 ml of heparinized medium and to- tal and differential cell counts performed.
cMean value for each group of five mice.
falling off. The peak macrophage response however does not occur until 48 - 72 hr after injection. This difference in kinetics of response allows one to examine selectively the ef-
fects of various agents on the inflammatory response of either the macrophage or polymorphonuclear leukocyte. By examining PHA-induced mouse peritoneal exudates at various times, we have been able to show that low molecular weight products from tu- mors (5) or from certain oncogenic murine viruses depress mac- rophage accumulation but have no effect on PMN accumulation (6).
A representative experiment is illustrated in Table III.
V. CRITICAL COJyiMENTS
A. Other Accumulation Assays
While we have described induction of peritoneal inflammatory reactions in mice and guinea pigs, they have also been elicited in rats (7, 8), hamsters (8), chickens (9), and rabbits (10).
Inflammatory reactions can also be quantified in the pleural cavity (7, 11) or in the lungs (12).
XI. MONONUCLEAR PHAGOCYTES IN VIVO 965 TABLE III. Inhibition of Macrophage (M0) Accumulation by Low Molecular Weight Viral Products3
Virus extract** Accumulated M0C Inhibition" Significance6
(ng) (xlO6 ± SE) (%) (p)
RLV 200.0
20.0 2.0 0.2
Buffer alone FLV
1200.0 120.0 12.0
1.2
Buffer alone MoLV
200.0 20.0 2.0 0.2
Buffer alone MMTV
720.0 72.0 7.2 0.7
2.2 1.8 1.6 2.8 4.2
2.1 2.8 3.0 3.7 5.2
3.0 4.8 1.9 1.7 7.2
5.8 5.2 5.6 4.9
± 0.1
± 0.4
± 0.2
± 0.2
± 0.3
± 0.3
± 0.4
± 0.5
± 0.1
± 0.4
± 0.2
± 0.2
± 0.2
± 0.2
± 0.4
± 0.6
± 0.6
± 0.8
± 1.0
48 57 62 33
<0.01
<0.01
<0.01
<0.02
60 46 42 29
<0.01
<0.01
<0.01
<0.02
58 33 74 76
<0.01
<0.01
<0.01
<0.01
0 0 0 2
NS NS NS NS Buffer alone 5.0 ± 0.5
Taken with permission from ref. 6.
bLow molecular weight virus extracts were injected subcu- taneously in 0.2 ml volumes into the thighs of five mice and macrophage accumulation determined.
cThe number of accumulated macrophages was determined by subtracting the number of macrophages (^1.8 x 10^) present in the peritoneal cavities of unstimulated mice from the number of macrophages present in the peritoneal cavities of PHA stimu- lated mice.
"Percentage inhibition of macrophage accumulation was cal- culated as % Inhibition = control - experimental
control
eAs determined by Student's t test.
^Not significant.
100
B. Mternate Methods
In vivo accumulation of inflammatory cells can also be measured by placement of nitrocellulose filters (7, 13) or glass coverslips (14) either subcutaneously or in the perito- neal cavity. The filters or coverslips can be removed at various times, stained, and the types and numbers of cells de- termined. Another technique is to label the bone marrow pre- cursor cells by giving the animals a pulse of [ H]thymidine.
The influx of cells into inflammatory sites (15, 16) or onto nitrocellulose filters or glass coverslips can then be fol- lowed using scintillation spectrophotometry. We have used this technique with mouse peritoneal exudate cells and found excellent correlation at 48 hr after injection between the amount of cell-associated radiolabel and the numbers of mac- rophages (16). The disadvantages of such techniques are that a considerable amount of label would be expected to be asso- ciated with PMN at the earlier times and considerable amounts of radiolabeled thymidine would be required for animals larger than mice or for large experiments. The method also requires two additional injections to each animal that may be undesir- able under certain circumstances.
Other methods that have been used include the air enclave method, in which a pocket is formed by injecting air subcu- taneously (17), or skin window techniques, in which an area of skin is exposed and then covered by a glass coverslip (18).
The coverslip is removed after various times, stained, and the numbers and types of cells determined. The former technique suffers due to the inherent variability associated with the formation of the air pocket and the latter requires micro- scopic examination of adhered cells from a large number of fields to reduce sampling errors.
REFERENCES
1. A. E. Postlethwaite and R. Snyderman. Characterization of chemotactic activity produced in vivo by a cell-mediated immune reaction in the guinea pig. J. Immunol. 114: 274- 278, 1975.
2. M. S. Hausman, R. Snyderman, and S. E. Mergenhagen. Hu- moral mediators of chemotaxis of mononuclear leukocytes.
J. Infect. Dis. 124: 595-602, 1972.
3. A. E. Postlethwaite, A. S. Townes, and A. H. Kang. Charac- terization of macrophage migration inhibitory factor ac- tivity produced in vivo by a cell-mediated immune reaction in the guinea pig. J. Immunol. 117: 1716-1720, 1976.
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Effects of neoplasms on inflammation: Depression of mac- rophage accumulation following tumor implantation. J. Im- munol. 116: 585-589, 1976.
5. G. J. Cianciolo, R. B. Herberman, and R. Snyderman. De- pression of murine macrophage accumulation by low molecular weight factors derived from spontaneous mammary carcinomas.
J. Natl. Cancer Inst. 65: 829-834, 1980.
6. G. J. Cianciolo, T. J. Matthews, D. P. Bolognesi, and R. Snyderman. Macrophage accumulation in mice is inhibited by low molecular weight products from murine leukemia vi- ruses. J. Immunol. 124: 2900-2905, 1980.
7. S. J. Normann and J. Cornelius. Concurrent depression of tumor macrophage infiltration and systemic inflammation by progressive cancer growth. Cancer Res. 38: 3453-3459, 1978.
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