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Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation ...
Journal

of Leukocyte

Biology

49:592-598

(1991)

Effect of Dietary Fish Oil on Development and Selected Functions of Murine Inflammatory Macrophages Neil Department

E. Hubbard,

Scott

D. Somers,

and

Kent

L. Erickson

of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis (N.E.H., Immunology, James N. Gamble Institute of Medical Research, Cincinnati (S.D.S.), Ohio

K.L.E.),

Division

of

Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation with interferon-y (IFN’y)and lipopolysaccharide due to an altered responsiveness to IFN-y. In an effort to elucidate further how dietary MFO effects macrophage function, we have studied the maturation of inflammatory macrophages from mice fed MFO compared with mice fed safflower oil(SF0) using several processes that serve as markers of the activational state. No significant differences in the recruitment or percentage of peritoneal exudate cells as macrophages after thioglycollate injection and no differences in spreading, binding, or phagocytosis of sheep erythrocytes or phagocytosis of yeast by inflammatory macrophages were observed when the dietary groups were compared. However, MFO macrophages had an altered capacity for peroxide release when stimulated with unopsonized zymosan (10-200 p.g/ml). Furthermore, to elucidate how MFO feeding could alter IFNyinduced responses of inflammatory macrophages, we assessed phorbol-12-myristate13-acetate-induced hydrogen peroxide production and expression of class II MHC determinants (Ia). There were no differences between macrophages from mice fed the two diets with respect to the production of peroxide when they were preincubated with 0.1-10 U/mI of IFNy. However, MFO macrophages had greater peroxide production after enhancement with 100 U/mI of IFNy. With respect to Ia induction, the percentage of macrophages responding to IFN’y was not altered by diet, and there were no differences in expression of Ia induced by 24 hr exposure to IFN’y. Thus the differential effect of MFO compared with SF0 is probably mediated not by an alteration in the maturation of inflammatory macrophages but rather through the alteration of IFN-y-induced functions such as peroxide production. Key words:

eicosapentaenoic acid, docosahexaenoic tional state, Ia expression

INTRODUCTION Macrophages (M’) are a highly diverse population of cells that are capable of executing a number of important biological functions and can be modulated by various cytokines, lymphokines, microbial factors, or lipid-based mediators [2,26,39]. For example, M can acquire the capacity to kill tumor cells when treated in vitro with interferon--y (IFNy) and lipopolysaccharide (LPS) [2]. The capacity to kill tumor cells requires first the ability to recognize and capture the target cells by binding to them and, second, the ability to release cytotoxic substances such as reactive oxygen intermediates (ROI) [8,32]. Macrophages can also be activated to initiate an immune response by processing antigen for presentation to T cells. This involves, at minimum, the expression of class II MHC determinants on the plasma membrane and can be induced by treatment with agents such as IFN-y or interleukin-4 (IL-4) [5]. Both activation for cytolytic capacity and activation for antigen presentation by the M4 as well as other functions may be down-regulated by

© 1991 Wiley-Liss,Inc.

acid, peroxide

production,

activa-

prostaglandin E2 (PGE2) [13,29,36,38], a biological mediator that can be altered by diet. Recently, much evidence has accumulated concerning the modulation of disease states by dietary fat. For example, diets high in vegetable oils that contain linoleic acid can promote the growth [1,10,17] and metastasis [15] of rodent mammary tumors. Diets high in marine fish oils can attenuate the growth of tumors [20] and can also affect inflammation and immunity [9,21,22,27]. The mechanism(s) involved in dietary fat modulation of disease is not well understood but most studies focus on alteration of eicosanoid (PGE, leukotrienes, and hydroxy fatty acids) production. Since eicosanoids can be involved in regulating macrophage function and activation, that cell becomes a focal point in dietary fat studies on immune response. We have begun to assess the effects of Received

July

25.

1990:

accepted

September

26.

1990.

Reprint requests: Neil E. Hubbard, Department of Cell Biology and Human Anatomy. School of Medicine. University of California, Davis, CA 95616.

Alteration

dietary fish oil on M taken from sites of sterile inflammation. Recently we reported that the activation of murine peritoneal M4i to kill tumor cells could be significantly altered by dietary fish oil [31]. M from mice fed a diet containing 10% menhaden fish oil killed fewer P815 mastocytoma cells upon in vitro activation with IFN’y and LPS compared with M4 from mice fed a diet containing 10% safflower oil (SF0). When activated pharmacologically, MFO M were equally competent for tumoricidal capacity, suggesting that the responsiveness of M to IFN-y was lowered by MFO feeding. Addition of indomethacin during activation with IFNy and LPS had no effect on tumoricidal activity. That evidence suggests a mechanism of MFO-altered immune response that is independent of modified eicosanoid production. In this report, our goal was to characterize what functional properties were altered by MFO feeding by studying several processes that serve as markers of activation [18] and to assess further the responsiveness of MFO M to IFN-y by studying the activation for enhanced peroxide production and for expression of Ia determinants; two processes sensitive to IFN’y. We show that MFO-feeding does not effect the in vivo maturation of M4, but we do provide further evidence that MFO alters the responsiveness in vitro of M to IFN’y.

MATERIALS AND METHODS Diets and Animals Female C57BL/6NCr mice (5-6 weeks old) were obtained from Charles Rivers (Kingston, NY). Animals were housed in autoclaved cages in a laminar flow hood and given sterilized water to minimize “spontaneous” activation of macrophages [25]. Mice were then fed purified diets for at least 4 weeks before experiments were performed. The diets tested were adequate in all nutrients and varied only in the type of oil fed, i.e.,either SF0 or MFO, which made up 10% of the diet by weight. Fatty acid composition of the oils used in the diets has been previously reported [7]. The basic composition of the semipurified diets was casein, 20%; DL-methionine, 0.3%; corn starch, 15%; sucrose, 44%; cellulose, 5%; AIN-76 mineral mix, 3.5%; AIN-76 vitamin mix, 2%; choline chloride, 0.2%; and SF0, 10% (California Oils, Richmond, CA); or MFO, 10% (Zapata-Haynie, Reedville, VA). The powdered diets were mixed and stored at -20%#{176}C under N,. Food and water were available ad libitum. Neither body weights nor food intake differed significantly (P > 0.05) between the two diets (data not shown).

Macrophages Cultures of thioglycollate (TG)-elicited peritoneal M4 were prepared as previously described [31]. Briefly, 3 days prior to use, mice were injected with 2.0 ml sterile

of Macrophage

Function

by Dietary

Fish

Oil

593

fluid thioglycollate broth prepared to manufacturer’s specifications (Difco, Detroit, MI). Peritoneal exudate cells (PEC) were harvested with Hank’s balanced salt solution (HBSS) (M.A. Bioproducts, Walkersville, MD), centrifuged at 500g for 5 mm, and resuspended in Eagle’s minimal essential medium (EMEM) (M.A. Bioproducts) containing 5% heat-inactivated fetalcalf serum (FCS) (Hyclone, Logan, UT), 1% L-glutamine, and 5 pg/ml gentamicin. M concentrations were adjusted after differential staining. There were no differences in the percentage of M4 elicitedfrom each group; M made up 85% of the PEC population. Following 90 mm of adherence in the tissue culture wells or plates, nonadherent cells were removed by vigorous rinsing with HBSS. Macrophages made up >95% of the final adherent cell population as judged by EAigG phagocytosis. There were no differences in the adherence efficiency between the dietary groups. Serum endotoxin level was 0.05) differences were observed in either the rate of uptake or the amount at each time point of phagocytosed sE between the two populations of M. Likewise, no significant differences between the diets in the percent of yeast postive M4’ after 15, 30, and 60 mm exposure were observed (Fig. 2). When M were stimulated with unopsonized zymosan, a significant difference in production of peroxide was observed between the diet groups (Fig. 3). SF0 M4 responded to increasing doses of zymosan with greater production of peroxide compared with MFO M4i. However, no differences were observed when MFO and SF0 macrophages were exposed to various doses of PMA (Fig. 4). Thus, using these markers of development, we find that MFO M differ from SF0 M only with respect to zymosan stimulation of peroxide production.

IFN’y-Enhanced

Peroxide

Production

Although we have observed little difference with respect to MFO and SF0 on the in vivo development of select markers of inflammatory M4, a reduced responsiveness to IFNy with respect to in vitro activation for cytolysis was always observed [31]. Therefore, to establish further how MFO may induce hyporesponsiveness to IFN-y, enhancement of peroxide production or expression of Ia was measured. Peroxide production is not constitutive in inflammatory M4 and so was stimulated by addition of PMA. To study the effects of diet on IFN-y-enhanced peroxide production, we incubated MFO and SF0 M for 4 hr with IFN-y before stimulation with 100 ng/ml PMA. Whereas MFO and SF0 M produced similar quantities of peroxide at low concentrations of IFN-y (0.1 and 1 U/ml), SF0 M peaked with respect to production at 10 U/ml while MFO M continued to release more peroxide at 100 U/mI IFN-y (Fig. 5). The ED50 (effective dosage at 50% enhancement) of IFNy for peroxide enhancement for SF0 M was 0.42 U/mI and for MFO M4 it was 4.2 U/ml.

Ia Antigen

Expression

Responsive peritoneal M4 do not constitutively express Ia antigen but acquire it by exposure to agents such as IFNy [5,12,34]. After a 24 hr exposure to logarithmically increasing levels of IFN’y, M from mice fed the two diets did not differ in their expression of Ia antigen (Fig. 6). Likewise, no differences were observed after 36 or 48 hr exposure to IFN-y (data not shown). Exposure to IFN”y for shorter time periods was not studied, because significant Ia antigen induction could not be observed.

of Macrophage

Function

by Dietary

Fish

595

Oil

5.

-0-

MFO

-e-

SF0

4.

sE/Me 3,

2’

.

,.......

20

0

40

60

TIME

80

100

(mm)

Fig. 1. Effect of dietary fat on the phagocytosis of opsonized SE by M. TG-elicited M4 were incubated with radiolabeled, opsonized SE for the times indicated. Phagocytosis was assessed by determining the level of radioactivity. There were no significant (P > 0.05) differences in the rate of SE phagocytosis when the two diet groups were compared. Results represent the mean ± SEM. 80 MFO SF0 60

%

Positive

40

20

A

-I 15

30

TIME

60

(mm)

Fig. 2. Effect of dietary fat on yeast phagocytosis. Heat-killed yeast were resuspended in media at a concentration of 1.5 x 107/ml then added to Md for the times indicated. With microscopic examination, there were no significant (P> 0.05) differences in the percentage of macrophages judged as positive for yeast phagocytosis between the diets at all three time points.

The percentage of Ia-positive cells was not altered (data not shown).

by diet

DISCUSSION We have development

assessed the effects of dietary fish oil on the of TO-elicited peritoneal M using pro-

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Hubbard

et al.

300

100 90 80

MFO

--

200 -..-

nM Peroxide!

mg

SF0

-I-

MFO

-C-

SF0

70

protein/I-Jr

nM Peroxide! mg protein/Hr

60

100

50 40 30 0

50

100 Zymosan

150

200

0.1

(tg/ml)

1

80

10

100

(U/mI)

IFN-1

Fig. 3. Effect of dietary fat on peroxide production stimulated with unopsonized zymosan. The amount of peroxide produced by MFO Md in response to 100 and 200 g/ml zymosan was significantly (P < 0.05) decreased compared with production by SF0 Md stimulated with the same levels of zymosan. Results are representative of at least three experiments.

-0-

0

Fig. 5. Effect of IFNy on the enhancement of peroxide production in Md from mice fed either MFO or SF0. IFNy did not enhance peroxide production in SF0 Md above 10 U/mI but did enhance production in MFO Md up to 100 U/mI. The ED for IFNy was calculated using log graph paper and in MFO Md was 4.2 U/mI and for SF0 Md it was 0.42 U/mI. Thus, a tenfold greater amount of IFNy was required for the 50% enhancement of peroxide production in Md from mice fed MFO compared with Md from mice fed SF0. These results are representative of three experiments.

SAF

60 600

nM

Peroxide!

mg

protein/Hr

500 40 400 20

OD410x

1O3

-0-

MFO

+

SF0

300

200 0.0

0.5

1.0

1.5

2.0 100

LOG

PMA

(nM)

Fig. 4. Effect of various doses of PMA on peroxide production by MFO and SF0 macrophages. Macrophages were incubated with various doses of PMA for 1 hr. Peroxide was analyzed as described in Materials and Methods. There were no significant (P > 0.05) differences in peroxide production at any dose.

cesses that serve as markers of activational state [18] and on capacities that require IFNy for enhancement (peroxide production) or induction (Ia antigen expression). Results from the present study allow us to conclude that M from mice fed a diet containing 10% MFO do not significantly differ developmentally from M of mice fed SF0. Murine M4 in various stages of activation, induced either in vitro or in vivo, can be characterized by their expression of several objective markers [18]. For example, resident peritoneal M are relatively incapable of EIgG or EIgM-C phagocytosis, spreading, or secreting

0 0

0.1

1 IFN1

10

100

(u/mi)

Fig. 6. Effect of dietary fat on Ia antigen expression in mouse peritoneal macrophages. TG-elicited macrophages were plated and exposed to IFNy at the doses indicated for 24 hr. Monolayers were fixed and assessed for Ia antigen expression as described in Materials and Methods. These results are representative of four separate experiments.

plasminogen activator, whereas in vivo activated M are quite capable of those functions [18]. On the other hand, resident M have a relatively increased content of 5’-nucleotidase and a high capacity for eicosanoid release [31] compared with more activated M4. TO brothelicited macrophages, which must be treated in vitro to

Alterationof Macrophage Function by Dietary Fish Oil become activated for tumor cell kill,have a higher level of surface receptor for mannose-containing glycoproteins than do in vivo activated macrophages, which are competent for tumor cell kill [16]. Thus, within the past decade, methods have evolved that enable investigators to differentiate activational states of M. There is no evidence to date that the activational state of the M4 can be modified by dietary fat. However, we have recently shown that a fish oil diet can significantly alter the in vitro responsiveness of inflammatory M4 to IFN-y [311. In that study, M4i from MFO-fed mice had a significantly reduced capacity to kill P815 mastocytoma cells when activated in vitro with physiological levels of IFN-y and LPS. Thus we suggested that M4i from MFO-fed mice were hyporesponsive to IFNy. Using several of the markers listed above, we found that MFO M did not significantly differ developmentally from SF0 M4. However, we did see a significant difference in the responsiveness of MFO M4 to zymosan with respect to peroxide production compared with SF0 M4. Zymosan can bind to mannose and CR3 receptors on M [11]. However, we cannot conclude that MFO has an effect on the levels of these receptors, because we saw no differences in the phagocytosis of yeast particles. The explanation for this may involve the postreceptor mechanism of zymosan-stimulated peroxide production. Recent evidence suggests that NADPH-oxidase, an enzyme necessary for ROI production, can be stimulated by arachidonic acid, which is released from M4 phospholipids in response to zymosan [23]. Since it has been shown that fish oil feeding decreases arachidonic acid levels in M phospholipids [6,7,24], decreased peroxide production by MFO M in response to zymosan may be due to decreased levels of released arachidonic acid that may act as a second signal for protein kinase C (PkC) or NADPH oxidase. The effect of fish oil is probably not due to a defect in PkC or NADPH oxidase, because there is no difference between the diets in peroxide production stimulated with various doses of PMA. We have looked at the effect of MFO feeding on the elaboration of cytolytic mediators such as R0I and proteins such as TNFa by M. The effects on TNFa secretion by M4 will be published elsewhere (S.D. Somers, and K.L. Erickson, submitted for publication). MFO and SF0 M appear to respond differently to higher levels of IFN-y with respect to peroxide production. The mechanism(s) for altered responsiveness to IFN-y is not known, however, as the behavior of many membrane proteins may be altered by changes in the lipid microenvironment [for review, see 30]. MFO feeding and the resultant fatty acid alteration of the M membrane may possibly influence behavior of the IFNy receptor and signal transduction mechanisms. Little is known concerning the signal transduction mechanism(s) for IFN-y; however, three separate events have been

597

observed. Upon occupation of a cell surface receptor, IFN-y stimulates a rapid (within 60 sec) alkalinization of the cytosol and a concomitant, rapid influx of 22Na [28]. These changes appeared to be important for mediating some of the subsequent genomic responses to IFNy [28]. IFN-y also stimulates a slow (10-15 mm) increase in efflux of 45Ca from prelabeled M [33] and also potentiates, but does not activate, PkC [3,14]. PkC probably plays a role in ROl production by M4 by stimulating the activity of NADPH-oxidase [37]. With respect to our results in the peroxide experiments, altered responsiveness of MFO M4 in IFN-y probably does not involve an alteration of PMA-stimulated PkC activity because the total amount of peroxide production was not affected by fish oil feeding; any effect on PkC would translate to a lesser or greater degree of peroxide production. We have begun to assess the activational state of M from mice fed different diets and show that, for the most part, there does not seem to be much of a difference between MFO and SF0 M. However, there appears to be some effect of diet on zymosan-stimulated peroxide production. Our recent report [31], however, has suggested that dietary fish oil may affect M4 function. Experiments that will extend and continue to clarify mechanisms associated with MFO effects on M function are currently underway.

ACKNOWLEDGMENTS The authors thank Dave Hoffsten of Adams Vegetable Oils (Woodland, CA) and A.P. Bimbo of Zapata-Haynie (Reedville, VA) for their generous contributions of the safflower oil and menhaden fish oil, respectively. This work was supported by NCI grant CA 47050.

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