DEPLETION OF MONONUCLEAR PHAGOCYTES:
PITFALLS IN THE USE OF CARBONYL IRON, CARRAGEENAN, SILICA, TRYPAN BLUE, OR ANTIMONONUCLEAR PHAGOCYTE SERUM
Paul A. LeBlanc Stephen W. Russell
INTRODUCTION
Selective depletion has been used to characterize the biologic functions of a number of different cell types, most notably lymphocytes. The approach has proved to be especially instructive when the function of a depleted population has then been reconstituted by adding back cells of the enriched subpopulation that is under investigation.
Selective depletion, by definition, works best when a unique characteristic of the cell of interest can be exploited.
This approach has recently become relatively straightforward for lymphocytes, because these cells are now known either to follow distinct differentiation pathways that can be inter- rupted or to express specific antigens that can be targeted, resulting in the destruction of a single subpopulation. In- formation that would suggest different pathways of differen- tiation or antigenic markers for individual subsets has yet to be obtained for mononuclear phagocytes. The means used to
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deplete mononuclear phagocytes have, therefore, been plagued by a common difficulty, namely, lack of specificity. The brief descriptions that follow consider the most commonly used
approaches.
II. CARBONYL IRON A. Introduction
Removal of mononuclear phagocytes with carbonyl iron theo- retically depends on phagocytic uptake of the iron, after which iron-containing cells are removed in a magnetic field. The method was first described by Rous and Beard (1) for the isola- tion of Kupffer cells from perfusate of rabbit liver. The car- bonyl iron approach has not proved useful for depletion of mo- nonuclear phagocytes in vivo.
5. Reagents
Carbonyl iron in powder form (General Aniline and Film Co., New York, New York) is used.
C. Method
Cells (10 /ml) are incubated with carbonyl iron (4 mg/ml) for 1 hr at 37°C with gentle agitation every 15 min. At the end of the incubation period a magnet is applied to the side of the tube for 1 min. The cells in suspension are gently withdrawn with a pipette to a new tube and the magnet reap- plied. This cycle is repeated five times.
D. Cri tical Comments
Carbonyl iron has been found to remove up to 98% of the macrophages from thioglycollate-elicited peritoneal cells (2).
However, the overall recovery of cells in these studies was low. Such low recoveries are a major problem with the tech- nique, as iron particles often are entrapped in clumps of cells or adhere to the surfaces of nonphagocytic cells. Thus, non- phagocytic, as well as phagocytic, cells may be nonspecifically lost. In addition, phagocytic cells other than mononuclear phagocytes, e.g., neutrophils (3, 4), will be removed. Finally, immature mononuclear phagocytes may not be avidly phagocytic and may not, therefore, be subject to removal by this technique.
III. CARRAGEENAN
A. Introduction
Carrageenan was first described as a mononuclear phagocyte toxin by Allison et al. (5). The toxic effect of carrageenan was ascribed by these authors to destabilization of the mem- branes of secondary lysosomes, resulting in release of hydro- lytic enzymes into the cytoplasm of treated cells.
B. Reagent
Carrageenan (Marine Colloids, Inc., Rockland, Maine) is used. The lambda form of carrageenan appears in several re- ports to be the most potent macrophage toxin, compared to other
forms of this material (6, 7). The reagent is dissolved ( 1 - 2 mg/ml) in phosphate-buffered saline by heating to boil- ing. The final preparation may be sterilized by autoclaving.
C. Method
1. Use in Vivo (6)
The usual routes of administration are intravenous or in- traperitoneal. The intravenous dose is 0.5 ml of a 1 mg/ml solution of carrageenan. For intraperitoneal use, the recom- mended dose is generally higher, i.e., 3 to 5 mg/mouse. The choice of route is based largely on whether a systemic or local, e.g., intraperitoneal, effect is desired. Substantial effects are seen during the first 48 hr after carrageenan is adminis- tered.
2. Use in Vitro (5)
Effective doses have varied from 25 yg/ml to 1 mg/ml; how- ever, the most used concentration has been 100 pg/ml, in con- junction with 24-hr preincubation.
D. Critical Comments
Carrageenan has a broad spectrum of effects apart from its suppression of mononuclear phagocytes. For example, it can have either a depressive or adjuvant effect on antibody produc- tion to either cellular or soluble antigens (6, 8, 9). The dif-
ferent forms of carrageenan have suppressive effects on cyto- toxic T cells that have been reported to vary independently of their antimacrophage effects (7). Carrageenan initiates the cleavage of Hageman factor and triggers the complement sequence by the alternative pathway (10). Carrageenan also binds to Cla, C2, and C4 (11). Its effects in vivo are so diverse that, rather than deplete macrophages, some investigators have used this agent successfully to induce inflammatory reactions that are rich in mononuclear phagocytes, i.e., so-called carragee- nan granulomas (12). In light of these facts, carrageenan must be viewed as one of the least specific of macrophage inhibitors.
IV. SILICA
A. Introduction
The antimacrophage effects of silica were also first des- cribed by Allison et al. (5), as part of an investigation of the fibrogenic properties of this particulate compound. Toxi- city for phagocytic cells was thought to stem from the induc- tion of membrane destabilization and rupture of secondary lyso- somes.
B. Reagent
Silica, quartz form, with a particle size of 1 - 5 ym (Whittaker, Clarke, and Daniels, Inc., White Plains, New York) is used. Surface contamination with iron oxide is removed by repeated boiling in 1 N hydrochloric acid until no further color change (green) is detectable. The cleansed particulate material is washed extensively (no further chloride reaction) in distilled water and then dry-heat sterilized for storage.
For use, the agent is suspended in tissue culture medium, after which clumps are dispersed by sonication. Suspensions should be made freshly for each use.
C. Method
1. Use in Vivo (13, 14)
The silica suspension should be sonicated immediately be- fore use to prevent possible embolism due to intravenously in- jected clumps. The dose of silica will vary batch to batch;
however, a useful starting range for mice has been from 1 to 40 mg. Both single and divided doses have proved effective.
2. Use in Vitro (15)
Silica for use in vitro should be suspended in serum- containing medium. This maneuver tends to reduce the direct toxicity of silica for cells. The dose used in culture is usually 50 - 100 yg/ml with cells at a population density of 10"/ml. Effects are seen after overnight incubation at 37°C.
D. Critical Comments
There are two major difficulties with silica. First, many of the same criticisms previously lodged against carrageenan apply equally to silica. For example, adverse effects on other cell types, most notably lymphocytes (16, 17), have been re- ported. Second, in many experiments conducted in vivo, effica- cious levels of silica have proved lethal to a portion of the animals injected. Results obtained from survivors of such a treatment would have to be treated with reservations. Finally, different batches of silica may have varying levels of anti- mononuclear phagocyte activity, even to the extent of lacking activity altogether (15).
V. TRYPAN BLUE DYE A. Introduction
Trypan blue has been reported to interfere with the rejec- tion of allogeneic skin (18) and tumors (19), as well as the elimination of antigenic, syngeneic neoplasms (20). Treatment of animals with the dye has also resulted in reduced resistance to Trypanosoma musculi (21) . Because of these, and other find- ings, it has been suggested that trypan blue may be a selective inhibitor of macrophage function (18). While there is little doubt that this agent affects macrophages, as will be seen from the comments in Section V.D, it has a wide spectrum of effects and should not, therefore, be viewed as selectively affecting macrophages.
B. Reagent
Trypan blue can be obtained either as a solution from a variety of vendors of tissue culture supplies or as a powder
(Sigma, St. Louis, Missouri). All preparations should be dialyzed exhaustively against distilled water to remove impur-
ities (19). . The dye is then lyophilized to dryness. For use, the powdered dye can be resolubilized in an appropriate buffer and sterilized by autoclaving.
C. Methods
1. Use In Vivo
Short-term suppression of macrophage function has been re- ported using a single injection of 1 mg/mouse (22). Sustained effects have been produced by Hibbs (19), using a loading dose of 4 mg/mouse and a biweekly maintenance dose of 1 mg/mouse, thereafter. The route of injection for the loading dose is in- traperitoneal, while the maintenance dose is given subcutane- ously.
2. Use In Vitro
A concentration of 1 mg/ml, using a 2- to 4-hr period of pretreatment, has proved effective (22).
D. Critical Comments
The mechanism by which trypan blue exerts its effects is not known. It is an inhibitor of certain hydrolytic lysosomal enzymes (23)· In addition, however, it inhibits fibrinolysis
(24) and the activation of plasminogen (24) , and interferes with the functions of a number of other proteins, such as anti- body and most of the components of the complement system (25).
This broad spectrum of activities has led one group of investi- gators to suggest that trypan blue mediates many of its biologic effects through a general affinity for proteins (25). Clearly, the activity or activities of this dye should not be considered specific for macrophages or macrophage-related proteins·
VI. ANTIMONONUCLEAR PHAGOCYTE SERUM Ä. Introduction
Of all of the methods that are currently available for ef- fecting the depletion of mononuclear phagocytes, conventional antiserum or monoclonal antibody raised against this cell type offers the greatest potential for achieving true specificity.
Even here, however, there are serious problems that center on cross-reactivity or sharing of antigens with other cell types.
B. Reagent
Either conventional antisera or monoclonal antibodies can be raised against mononuclear phagocytes.
1. Conventional Antiserum
Antimononuclear phagocyte serum can be produced in any con- venient laboratory animal species. Xenogeneic antiserum is used as there are no allotypic markers yet defined for mononu- clear phagocytes. Every effort should be made to enrich the immunizing cell population for mononuclear phagocytes. Exten- sive washing of an adherent population is the most commonly used approach to enrichment. The protocol for immunization has varied from investigator to investigator. For use in vivo in mice, we have found "early" IgG produced in rabbits to be most effective at depleting mononuclear phagocytes. Immuniza- tion is started with a single, intramuscular injection of cells emulsified in Freund1s complete adjuvant. This injection is followed by two intravenous administrations of cells suspended in saline, given at weekly intervals. Serum is harvested 1 week later and fractionated to yield the IgG fraction. Extensive absorption of the antiserum is then required to render it spe- cific.
2. Monoclonal Antibody
The standard hybridization technique (26) can be used to produce the clones. If antibody against mouse mononuclear pha- gocytes is desired, rats can be immunized. The rat spleen cells are then fused to mouse myeloma cells. The fusion effi- ciency and stability of the resulting interspecies hybrids have been good; however, the need for a HAT sensitive rat myeloma line suitable for rat-rat fusions is clear. At this writing there is the expectation that such a line will be available soon. If antibodies against the mononuclear phagocytes of other species are desired, mice can be immunized, allowing mouse - mouse hybrids to be used for the production of antibody. When an antibody of interest has been identified, it can be concen- trated from tissue culture supernate or produced in quantity in ascites fluid (27).
C. Methods
1. Use In Vi vo
An appropriate amount (0.1 - 0.5 ml) of the antiserum is injected. If recruitment of mononuclear phagocytes into a
lesion is to be interdicted, the route of administration should probably be intravenous. If local depletion is the aim, it may be preferrable to inject locally, for example, intraperitoneal- ly. Repeated administration will be necessary to sustain in vivo depletion.
2. Use In Vitro
For eliminating mononuclear phagocytes in vitro, the standard approach to producing antibody/complement-mediated cytotoxicity is followed.
D. Critical Comments
Achieving true specificity for mononuclear phagocytes with conventionally raised antisera is extremely difficult. The difficulty is caused by the fact that mononuclear phagocytes share antigens or bear antigens that cross-react with those of other cell types, e.g., neutrophils (28) and lymphocytes (27).
Claims for specificity attained after few or no absorptions are quite simply the result either of not looking at enough other cell types or not using an assay sensitive enough to de- tect cross-reactivity. In this latter regard, we have found assays such as antibody/complement-mediated cytotoxicity or immunofluorescence to be relatively insensitive. Assays that measure interference with the functions of cell types other than mononuclear phagocytes have proved to be much more sensi- tive indicators of nonspecific effects. As an example of how extensive the process of absorption must be, rabbit antiserum produced against murine bone marrow mononuclear phagocytes in our laboratory has routinely required 15 separate absorptions with packed mouse erythrocytes and absorption with an addi- tional 101 0 cultured mouse tumor cells before antispecies ac- tivity has been completely removed.
Many of the problems of nonspecificity might be overcome with the use of monoclonal antibodies, providing screening is adequate to eliminate those that bind to cell types other than mononuclear phagocytes. Monoclonal antibodies of interest are now being developed in several laboratories. When working with a single species of antibody that is directed against a single antigenic determinant, new problems may arise. Chief among these is that the conditions of cell - antibody interac- tion may simply not be adequate to effect depletion, either in vivo or in vitro. In such cases it might be necessary to use a "cocktail" consisting of several different monoclonal anti- bodies. There is also the potential problem of the antibody that will not fix complement. This may present no real diffi- culty in vivo because it is not clear that depletion in vivo
is effected by this means. If the density of antigenic deter- minants bound by a complement-fixing antibody is too low to permit cytolysis, or the antibody is not of the complement-
fixing type, then the addition of a "second layer" of comple- ment-fixing antibody to the system might serve to eliminate mononuclear phagocytes in vitro.
In either case, there is the problem of sustaining deple- tion in vivo. Because xenogeneic antibodies are employed, the recipient will respond with antibodies of its own that will be directed against the foreign protein(s). Immune elimination of the injected antibody could result. It may be possible to blunt or to circumvent completely the recipient's response if antibody is first deaggregated before it is injected. This ap- proach has been used successfully in the therapeutic application of antilymphocyte globulin (29), and is used routinely as a means of inducing tolerance in experimental animals (30). If such steps are not taken, the recipient's antibody response might neutralize the antimononuclear phagocyte globulin allowing the bone marrow to replenish the depleted population.
VII. CONCLUDING REMARKS
The state of the art is such that we simply cannot yet make the claim that an easy approach is available to the depletion of mononuclear phagocytes in vivo. Of the methods considered here, certainly antibody (especially monoclonal antibody) holds the greatest promise for the future. We are hopeful, given the current amount of interest in the field, that within the next few years a monoclonal antibody preparation that is suitable for depletion studies and that has absolute specificity for mononu- clear phagocytes will become available. Perhaps, then it will be possible to answer definitively those questions that stand unanswered today because of the problems of nonspecificity.
Acknowledgment
The authors would like to thank Ms. Brenda Brown for typing the manuscript. Supported by NIH Research Grant CA-31199 and NRSA CA-06088 and Contract CB-84271. S.W.R. is the recipient of Research Career Development Award CA 00497.
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