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SECRETORY FUNCTIONS OF MONONUCLEAR PHAGOCYTES:

OVERVIEW AND METHODS

FOR PREPARING CONDITIONED SUPERNATANTS

Philip Davies

INTRODUCTION

The utilization of well-controlled tissue culture condi- tions has led to the identification and characterization of a variety of secretory products synthesized by cells of the mononuclear phagocyte system. It is now apparent that the spectrum of products released by various mononuclear phago- cyte populations into their cuLture medium is markedly depen- dent on the source of the cells as well as the kind of stimuli to which they are exposed in vivo or in tissue culture. These variations depend on complex cellular regulatory mechanisms by which mononuclear phagocytes respond to their pericellular en- vironment. Such modulation is triggered in part through a number of plasma membrane receptors and high affinity binding sites that interact with various ligands in the pericellular environment. Such recognition leads to the initiation of a complex series of biochemical events, which among other things, induce and stimulate the secretory activity of the cells.

METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.

MONONUCLEAR PHAGOCYTES 5 4 9 AU rights of reproduction in any form reserved.

ISBN 0-12-044220-5

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II. SECRETORY PRODUCTS OF MONONUCLEAR PHAGOCYTES

It is not intended to provide an extensive account of these products here as this has been done elsewhere (1-3).

Table I provides a summary of some of the secretory products of mononuclear phagocytes. These include a large group of hydrolytic enzymes (for review see ref. 4), which can be dis- tinguished from each other by the conditions under which they are secreted. Lysozyme, released on a constitutive basis by mononuclear phagocytes, is little affected by the source of cells or the kinds of stimuli brought into their environment in vivo or in culture. On the other hand, the release of both neutral proteinases and lysosomal acid hydrolases is markedly dependent upon the environment from which the cells are obtain- ed and also the nature of the stimuli added to cultured cells.

While resident cells secrete minimal amounts of neutral pro- teinases, mononuclear phagocytes elicited by inflammatory stimuli secrete these enzymes in significant amounts over pe- riods of several days without further stimulation. Also, such secretion can be initiated in resident cells in culture by certain inflammatory stimuli, or enhanced further in elicited populations. Lysosomal acid hydrolases are secreted at a low rate in both resident and elicited populations (37). Such re-

TABLE J. Some Secretory Products of Mononuclear Phagocytes0

Product Reference Hydrolytic enzymes

Lysozyme (5) Collagenase (6) Elastase (7) Plasminogen activator (8)

Proteoglycan degrading enzyme (9)

Factor D (10) Neutral caseinase (11)

Lysosomal acid hydrolases (4) Arachidonic acid oxygénation products

Prostaglandin E2 (12) Prostaglandin F« (12) 6-Ketoprostaglandin F-* (13)

Thromboxane B~ (14) 12-Hydroxyeicosatetranoic acid (15)

SRS (16)

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TABLE J. (cont.) See also section by Humes Modulators of cellular behavior

Stimulant of stem cell proliferation (17)

Thymic maturation factor (18) T Lymphocyte mitogen (19) B Lymphocyte mitogen (20) Inhibitor of T lymphocyte proliferation (21)

Inhibitor of fibroblast proliferation (21) Stimulant of collagen synthesis (21)

Stimulant of angiogenesis (22) Antagonist of steroid induction of hepatic

phosphoenolpyruvate carboxykinase (23) Low molecular weight chemotactic

factor for polymorphonuclear leukocytes (24) Substances cytostatic or cytotoxic to infectious agents and eukaryotic cells

Interferon (25) Oxygen metabolites

Hydrogen peroxide (26) Hydroxyl radicals (27)

Superoxide (28) Listericidal factor (29)

Vitamin B12~binding protein (30)

Arginase (31) Cytotoxic proteinase (11)

Complement components

Clq (32) C2 (33) C3 (32) C4 (33) Factor B (34) Factor D (10) Others

Pyrogen (also known as interleukin 1) (35)

Tissue thromboplastin (36)

aThis table is not intended to provide detailed documenta- tion of the secretory products of mononuclear phagocytes.

Only one reference is given for each product. These do not

account fully for the different types of mononuclear phagocytes secreting that product; the reader is referred to the other

sections of this monograph dealing with specific products and to reviews of the subject referred to in the text for detailed information.

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lease can be greatly increased by introduction of certain types of phagocytic stimuli possessing chronic inflammatory potency such as antigen-antibody complexes or zymosan parti- cles. Such activity has also been ascribed to components of the complement system such as C3b (38) and C5a (39) as well as a number of other soluble substances that have the common property of being activators of the alternative pathway of the complement system (40). In contrast, phagocytic stimuli such as carbon particles, latex beads or erythrocytes, which lack chronic inflammatory potency, do not stimulate the selec- tive release of lysosomal hydrolases.

It is now established that mononuclear phagocytes from various sources synthesize a broad range of arachidonic acid oxygénation products (for review see ref. 41). Prostaglandin E2 and prostaglandin F2ot were the first such products to be discovered. Subsequently other products such as prostacyclin, thromboxane A2, as well as hydroxy fatty acid products of the lipoxygenase pathway have been described.

Recent studies in a number of laboratories (see chapter by Colten, this volume) have established that mononuclear phago- cytes secrete many of the components that constitute the alter- nate and classical pathways of the complement system.

Mononuclear phagocytes secrete diverse substances that affect the proliferative and differentiated functions of cells found in their immediate environment (see Table I). In many instances, the biochemical and physical properties of these substances have not been established. In some cases, the bio- logical activities by which they have been characterized may well be shared by one molecule. An example of this is the possible identity of a T lymphocyte-activating factor and py- rogen released by human peripheral blood monocytes (42). It is entirely possible that other macrophage secretory products identified by biological assay may be single molecular entities.

Since one of the primary functions of mononuclear phago- cytes is the mediation of host defense mechanisms, it is to be expected that several of the products that they release into their environment should inhibit the growth or be cytotoxic to a variety of infectious agents as well as eukaryotic cells

(Table I). Such molecules range from low molecular products of oxygen metabolism to macromolecules, such as interferon, and factors interfering with the growth of intracellular para- sites and bacteria.

We have already mentioned the proteinases secreted by mono- nuclear phagocytes; it is notable that these cells also secrete o^-macroglobulin (43), which is an inhibitor of these enzymes.

Also mononuclear phagocytes secrete a tissue factor-like mole- cule (36) which initiates the blood coagulation cascade. Since plasminogen activator has as its substrate the zymogen of the

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enzyme responsible for fibrin degradation, it is apparent that mononuclear phagocytes secrete products that can (a) trigger the activity of a humoral system, blood coagulation, (b) remove the product of its activation, fibrin, as well as (c) produce an endogenous inhibitor (a^-macroglobulin) of the numerous proteinases involved in the sequential steps leading to fibrin

formation.

The physiological and pathological significance of these secretory activities of mononuclear phagocytes remains to be established. Obviously the appropriate use of systems for mea- suring secretory activities of mononuclear phagocytes can aid greatly in answering such questions.

III. PREPARATION OF CONDITIONED MEDIUM

Since other contributors to this section will deal with specific details concerning individual products, we shall con- fine ourselves to general points concerning the requirements for maintaining mononuclear phagocytes in culture and preparing conditioned culture medium for measurement of products.

A. Monitoring Viability of the Cultures

An essential prerequisite for any systematic study of the secretory activity of mononuclear phagocytes is that the cul- tured cells be fully viable for the duration of the experiment.

This requirement can be ascertained in a number of ways. Cell cultures should be monitored microscopically, preferably by phase contrast optics, to ensure that cells show a morphology consistent with that expected of the cell population being studied. Any irregularity in the morphologic appearance of the cells immediately suggests potential problems of two kinds:

(a) that the donor of the cells has a pathological abnormality or an infection that is being expressed by direct effect on the cultured mononuclear phagocytes, or (b) that the procedures for preparing the cells have not been properly conducted.

Specific biochemical approaches can be used to monitor the viability of cell cultures.

Starting cell populations should be counted and adjusted to a constant number to give a regularly plating density. The pre- cise number of cells can be determined by measuring the DNA con- tent of the culture. This can be readily determined by the use of fluorometric methods such as that described by Setaro and Morley (44), which we have regularly utilized in the laboratory.

In primary cultures of macrophages, which do not normally show mitotic activity, the maintenance of DNA levels in cell cultures

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is a good indicator of the retention of cell viability, al- though it does not necessarily indicate full maintenance of cellular function. Protein content of cells of cultures thor- oughly washed in serum-free medium can also be determined by conventional techniques. We have also made a practice of es- timating the distribution of the cytoplasmic enzyme lactate dehydrogenase between cells and their culture medium. This assay is a particularly useful one when the effect on the secretory activities of mononuclear phagocyte cultures of stimuli of unknown cellular toxicity is examined. Several cautions need to be made in relation to measurements of this enzyme. The absence of elevated levels of lactate dehydro- genase in culture medium is not necessarily a definitive indi- cation of a lack of toxicity. This enzyme is relatively un- stable, being readily inactivated at low concentrations on glass and other surfaces, particularly in the absence of other proteins. (It is to be noted also that lactate dehydrogenase is destroyed by freezing.) Therefore, cellular levels of the enzyme should also be estimated with levels of enzyme equal to or greater than those at the beginning of the experiment being a reasonable indicator of cell viability.

In many instances, the rate of secretion of a given mono- nuclear phagocyte product is dependent on the in vivo environ- ment from which the cells are obtained. This has been parti- cularly well established in the case of mouse peritoneal mac- rophages. As has been described by Edelson and Duncan (this volume) 5'-nucleotidase and leucine aminopeptidase are useful

markers that can be readily assayed to indicate whether cells exhibit activities commensurate with the environment from which they are obtained. Further relevance can be given to these measurements when they are related to the DNA or protein contents of the cells.

In studies of secretory activities, it is particularly useful to be able to relate this to that of a constitutive secretory activity of mononuclear phagocytes, such as lysozyme.

Such measurement is useful not only for measuring constitutive secretory function coordinate with that of another product but also as a subsequent indicator of the functional viability of the cells after exposure to any given stimulus.

The assay of the parameters, described above at the be- ginning and during the course of an experiment during which secretion of a given product is determined, provides not only a baseline for quantitative measurement of the product but also an indicator of the viability of the cells during the course of the experiment.

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B. Preparation of Conditioned Media

One of the primary objectives of preparing conditioned, culture medium is that the secretory product be recovered in quantitative amounts. The difficulties encountered in achieving this objective are several: (a) instability of the secretory product in the culture medium; (b) degradation of the product by an enzyme present in the culture medium; and

(c) loss of activity due to inhibitors present in the culture medium.

The difficulties encountered in (a) can be overcome if the secretory product decomposes into a stable metabolite that can be quantitated, e.g., 6-ketoprostaglandin F^a is the stable metabolite of prostaglandin I2. Degradation of a secretory product by enzymes can be overcome by culturing cells in medium lacking sera or sera in which the enzyme has been in- activated. Difficulties with endogenous inhibitors can again be overcome by exclusion of serum or inactivation of inhibitor.

The acid treatment of serum to destroy endogenous inhibitors of neutral proteinases is a good example of such a procedure.

The inclusion of a radiolabeled exogenous standard can aid in monitoring the recovery of a secreted product from condi- tioned media. Spontaneous breakdown of a product can be mea- sured by inclusion of culture vessels containing medium only to which is added a known quantity of the secretory product being quantitated. Culture vessels containing medium only should always be included to account for background activity of the product being measured.

Acknowledgment

The excellent secretarial assistance of Mrs. Carolyn Kradjel is gratefully acknowledged.

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pp. 1273-1294. Martinus Nijhoff Bv, The Hague, 1980.

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30. M. Rachmilewitz and M. Schlesinger. Production and re- lease of transcobalamin II, a vitamin B12 transport protein by mouse peritoneal macrophages. Exp. Hematol.

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Ábra

Table I provides a summary of some of the secretory products  of mononuclear phagocytes
TABLE J. (cont.) See also section by Humes  Modulators of cellular behavior

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