Immunity in Mice Infected With Mycoplasma - Europe PMC

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Immunity in Mice Infected With Mycoplasma pulmonis or Given Carcinogens by Injection. MICHAEL BENNETT, MD,. RICARDO UAUY, MD, PhD, and. SCOTT M.
Dietary Fatty Acid Effects

on

T-Cell-Mediated

Immunity

in Mice Infected With Mycoplasma pulmonis or Given Carcinogens by Injection

MICHAEL BENNETT, MD, RICARDO UAUY, MD, PhD, and SCOTT M. GRUNDY, MD, PhD

From the Department ofPathology, Pediatrics, Internal Medicine, Biochemistry and Centerfor Human Nutrition, University of Texas Health Science Center at Dallas, Dallas, Texas

To test whether or not diets enriched in w-6 polyunsaturated fatty acids are significantly immunosuppressive, B10.D2, DBA/2, and C3B6F1 mice were fed diets enriched for fatty acids: linoleic (POLY), oleic (MONO), palmitic (SAT), or eicosapentanoic (FISH). The B10.D2 and DBA/2 mice were given injected methylcholanthrene several weeks later, and immune studies were performed several months after carcinogen treatment. In conventional quarters, DBA/2 fed the POLY diet survived poorly, and many were infected with Mycoplasma pulmonis, even if given the vehicle, tractinoin, only. B10.D2 mice survived well unless on the POLY diet and given methylcholanthrene. Nevertheless, only mice on the POLY diet were significantly immunosuppressed, and only T-cell -mediated cutaneous sensitivity reactions were affected. Antibody, natural killer cell, and natural cytotoxic cell re-

sponses were not influenced by the diets. The C3B6F1 mice were assessed for immune functions prior to carcinogen (ethylnitrosourea) instillation into the trachea, and no immunosuppression was detected. After instillation, mice on the POLY and MONO diets were suppressed for T-cell cutaneous responses. Deliberate infection with Mycoplasma pulmonis resulted in suppressed cutaneous T-cell responses in the POLY group of C3B6F1 mice, and aspirin partially reversed the immunosuppression. Mice on the FISH diet were resistant to immunosuppression. It is tentatively concluded that diets rich in w-6 polyunsaturated diets, while not directly immunosuppressive, do predispose animals to suppression of certain T-cell - mediated immune responses. This immunosuppression can be "triggered" by infection and/or by exposure to carcinogens. (AmJ Pathol 1987, 126:103-113)

THE PATHOGENESIS of both atherosclerosis and neoplastic disease may be affected by the quality and quantity of fatty acids in the diet. Polyunsaturated fatty acids of the w-6 variety, when substituted for saturated fatty acids, will lower plasma levels of total cholesterol and low density lipoproteins (LDLs). ' The w-3 polyunsaturated fatty acids cause a lowering of plasma triglycerides.2 Monounsaturated fatty acids have been considered to be neutral in their action on plasma levels of total cholesterol and LDL;3 but when they are substituted for saturated fatty acids, plasma LDL levels falls.4 Therefore, a major shift from saturated to w-6 or w-3 polyunsaturated or to monounsaturated fatty acids may reduce the risk for athero-

effects of diets enriched with w-6 fatty acids have been emphasized. Although factors such as total amount of fat and calories and the presence or absence of other dietary constituents may influence the importance of dietary fats, considerable evidence exists that diets rich in w-6 polyunsaturated fatty acids predispose to induction of cancer by chemical carcinogens.7-14

Supported by the Veterans Administration, Grants RR00890 and HL-29252 (NIH/HDS/DHHS), The Southwestern Medical Foundation, The Moss Heart Foundation, Dallas Texas, and Mrs. Estelle Watlington, Amarillo, Texas. Accepted for publication August 26, 1986. Address reprint requests to Scott M. Grundy, MD, PhD, Center for Human Nutrition, University of Texas Health Science Center at Dallas, Room G4-100-5323 Harry Hines Blvd., Dallas, TX 75235.

sclerotic disease. With respect to neoplastic diseases, diets high in dietary fat are associated with increased incidences of breast and colon cancers.5 6 The potentially harmful 103

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BENNETT ET AL

A major mechanism by which w-6 polyunsaturated fatty acids may predispose animals to neoplastic disease is suppression of immunocompetence.'5 These fatty acids can directly suppress immune functions in vitro,'6"17 and dietary w-6 polyunsaturated fatty acids can result in immunosuppression.'8120 Serum from mice fed such diets, especially the lipoprotein fraction, can inhibit mitogenic responses of lymphocytes in vitro. 10 Since linoleic acid is a precursor of arachidonic acid and therefore prostaglandins, the immunosuppressive effects of prostaglandins have been studied in detail.2'23 Still, other studies indicate that polyunsaturates are not necessarily immunosuppressive or that other factors may influence any immunosuppression detected.24 27 The purpose of the studies reported here was to determine whether or not dietary w-6 polyunsaturated fatty acids are more immunosuppressive than w-3 polyunsaturated and/or monounsaturated fatty acids, especially after injection of chemical carcinogens. The results of our studies suggest that the w-6 polyunsaturated fatty acids are indeed immunosuppressive of T-cell-mediated immune responses in vivo, but only under certain conditions. For example, mice apparently are not immunosuppressed unless they are infected with organisms, eg, Mycoplasma pulmonis, and/or are given injected carcinogens. We propose that the immunologic effects of dietary constituents cannot be accurately determined when animals are housed in "conventional" animal quarters in long-term studies. Specific pathogen-free (SPF) or germ-free facilities are almost mandatory. Nonetheless, under the influence of infection or chemical carcinogens, w-6 polyunsaturates are more immunosuppressive than monounsaturates and w-3 polyunsaturates. The mice fed the fish oil appeared to be quite resistant to immunosuppression.

Materials and Methods Mice Male (C3H X C57BL/6)FI (C3B6F1) and female DBA/2 and BIO.D2 mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). DBA/2 and BIO.D2 mice are both H-2d types, but DBA/2 mice are resistant and B I0.D2 mice are susceptible to induction of fibrosarcomas28 by methylcholanthrene (MC). The animals were maintained in conventional facilities and were placed on experimental diets at 7 weeks of age. The mice were cared for as prescribed in the Guide for the Care and Use ofLaboratory Animals, published by the National Institutes of Health.

AJP * January 1987

Diets Six diets were employed in two studies. Four diets contained 30% of calories as fat. The first was rich in saturated fatty acids (SAT) and contained palm oil as its only fat. The second was high in monounsaturated fatty acids (MONO) and contained only high-oleic safflower oil. The third contained only high-linoleic safflower oil and thus was rich in w-6 polyunsaturates (POLY). The fourth diet contained menhaden oil (FISH), which was rich in eicosapentaenoic acid, w-3 fatty acid. In the preparation of these three diets, fatfree diets (ICN Nutritional Biochemicals, Cleveland, Ohio) were enriched with one ofthe oils. The fifth diet was low in fat (LoFat) and contained 10% of calories as fat with equal quantities (1: 1: 1) of saturated, monounsaturated, and w-6 polyunsaturated fatty acids. The sixth diet was conventional Teklad diet (CHOW), which contains 10% of calories as fats equally divided between vegetables and animal fats. The mice were fed once daily and were allowed to eat ad libitum. One group of C3B6F I mice on the POLY diet received aspirin (acetylsalicylic acid) in the drinking water (50 mg/500 ml). The diets were supplemented with ICN vitamin diet fortification mixture. The contents (in grams per milogram) were: vitamin A acetate, 1.8; vitamin D2, 0.125; tocophero acetate, 22.0; ascorbic acid, 45.0; inositol, 5.0; choline chloride, 75.0; menadione, 2.25; para-amino benzoic acid, 5.0; niacin, 4.25; riboflavin, 1.0; pyridoxine HC 1, 1.0; thamine, 1.0; calcium pantothenate, 3.0; biotin, 0.02; folic acid, 0.09; and vitamin B,2, 0.00135.

Experimental Design The protocols are depicted in Figure 1. In the first experiment with B0.D2 and DBA/2 mice, groups of 50 mice of each strain were placed on the diets 6 weeks prior to injection of 0, 12.5, or 125 ,ug methylcholanthrene (Sigma Chemicals, St. Louis, Mo) in 0.05 ml trioctanoin (Eastman Kodak Co., Rochester, NY) subcutaneously. In the second experiment, C3B6F1 mice were given intratracheal injections of 600 ,g ethyl nitrosourea (Sigma) in 50 ,ul RPMI 1640 medium or medium only. A separate group of C3B6F1 mice were infected intratracheally with 4 X 1 8 Mycoplasma pulmonis organisms. Immune Studies Groups of 4 mice were immunized intraperitoneally with 5 X 108 sheep erythrocytes (SRBCs) and were bled on Days 5, 10, and 18. Serum samples were divided, and one-half were treated with 2-mercaptoeth-

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1. B10.D2 and DBA/2 female mice Diets Initiated

Methylcholanthrene

7

13

Immune Studies

Age (weeks)

I 41

II. C3B6F1 male mice

Ethylnitrosourea

Lung Histology

Immune Studies

I Diets Initiated

Immune Studies

8

24

Fat pad Mycoplasma Analysis pulmonis

Immune Studies

Age (weeks) 32

36

44

45

61

Figure 1- Experimental protocols.

anol (2ME) to inactivate IgM molecules. Serial twofold dilutions of antisera, 25 ,ul, were tested for the ability to agglutinate SRBCs.29 The values are expressed as log2 titers of IgG (2ME-resistant) or IgM

(2ME-susceptible) agglutinating antibody. Natural killer (NK) cell activity of spleen cells was determined by culturing various numbers of spleen cells with 2 X 104 51Cr-labeled YAC- 1 lymphoma cells (prototype NK cell target cells) for 4 hours at 37 C in volumes of 0.2 ml in triplicate. The isotope released into the supernatant fluid was counted with a Packard PRIAS gamma scintillation counter.30 Controls included maximum release (detergent treatment of YAC- 1 cells) and spontaneous release (no effector cells). The formula for percent specific cytotoxicity is 5ICr, cpm E - SR/MR - SRXIOO, where E = experimental, SR = spontaneous, and MR = maximum release, respectively. Since the mice were several months old, some were given intraperitoneal injections of 0.12 mg polyinosinic-polycytidylic acid (pI: pC) 1 day earlier to "boost" NK cell function.31 The in vivo measurement of NK and natural cytotoxic (NC) cell32 activities was performed by determining the ability of mice (groups of 4) to "clear" radiolabeled YAC- 1 (NK) or WEHI- 164.1 (prototype NC target) cells from their lungs.33 Briefly, 5'Cr-labeled YAC-1 and '251-iododeoxyuridine ('25IUdR) labeled WEHI- 164.1 cells (5 X 105 each) were infused intravenously, and the lungs were removed 4 hours later. The 5'Cr and 125I radioactivities were determined as above. The values are expressed as the percent retention of label (therefore, cells) in the lung.

The greater the NK/NC function, the lesser percent retention of label is observed. Cutaneous sensitivity (CS) to trinitrochlorobenzene (TNCB, Eastman Kodak Company) was one measure of T-cell - mediated immunity. Groups of 6 mice were "painted" with 50 A1 7% TNCB in 4: l acetone/olive oil on shaved abdominal skin to sensitize them. Five days later, the mice were challenged with 20 jul 1% TNCB on the left pinnae. The right pinnae were painted with the vehicle. An engineer's micrometer was used to measure the thickness of the ears 24, 48, and 72 hours after challenge.34 The values are expressed as the increase in ear thickness (A 10-3 inches) from time 0. Controls included mice sensitized but not challenged and mice challenged but not sensitized. A Student t test was performed to determine the significance of differences between geometric or arithmetic means. Delayed type hypersensitivity (DTH) to SRBC was determined by immunizing mice with 106 SRBCs intravenously. Five days later, the mice were challenged with 108 SRBCs/50,ul in the right footpad. A change in footpad thickness was measured 24 hours later. DTH to allogeneic spleen cells was determined by immunizing C3B6F1 mice with 5 X 106 DBA/2 spleen cells in the left footpad. Seven days later, the mice were challenged with 107 DBA/2 spleen cells in the same footpad. The change in thickness of the footpad was measured 24 hours later. Controls were similar to the one for CS to TNCB. The responsiveness of spleen cells to mitogens was determined by incubating 5 X 105 cells for 3 days with

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BENNETT ET AL

1.0 or 0.5 ,ug concanvalin A (Con A) or 1 0pg lipopolysaccharide (LPS) in volumes of 0.2 ml in flat-bottomed wells of microtiter plates (triplicate samples). Each well was pulsed with 0.5 uCi 3H-thymidine for the final 18 hours to harvest the cells. The values are expressed as the mean ± SEM blastogenesis (cpm 3H-thymidine, mitogen-medium control).

AJP o January 1987 0

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SAT

0-

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MONO POLY

lx

Analysis of Lipids in Fat Pads and Spleen Intraabdominal mesenteric fat pads and spleens were homogenized with the use of a Dounce homogenizer; lipids were extracted with the use of chloroform/methanol as partition solvent.35 Lipid samples were analyzed immediately or stored with BHT as antioxidant at -20 C for later chromatographic analysis. We used thin-layer chromatography (TLC) on silica gel G plates with a 250-, layer thickness with hexane/diethyl ether/methanol/acetic acid (80:20: 3:2) as a solvent system to separate triclycerides from adipose tissue and phospholipids from spleen, comparing their migration with known standards. Specific lipid bands from TLC were scraped and fatty acids methylated with the use of the direct transesterification method.36 Methyl esters of fatty acids were stored under nitrogen at -20 C prior to gas chromatography analysis. We performed separation and quantification of fatty acid methyl esters with flame-ionization detector gas chromatography in duplicate samples, comparing their retention time with authentic standards. A Hewlett Packard Model 5700 GC chromatographer equipped with a 0.75-mm bore 30-m capillary column filled with SP-2330 phase was used for analysis. The relative proportions of individual fatty acids were expressed as the percentage of total fatty acids greater than 12 carbons.

Analysis of Lung Lesions The C3B6Fl mice given intratracheal injections of ehthylnitrosourea were sacrificed 15 weeks later. The lungs were fixed in 10% buffered formalin, and the paraffin sections were cut so that we could obtain coronal sections of the lung. At least two sections per lung were examined after staining with hematoxylin and eosin.

Results Studies of B10.D2 and DBA/2 Female Mice B 10.D2 mice given the vehicle or 12.5 jug methylcholanthrene survived well, irrespective of diet (Figure 2). However, B 10.D2 mice on the POLY diet and given 125 ,ug of the carcinogen survived poorly.

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B1O.D2 VEHICLE Ia

O0

0

100 TIME AFTER ADMINISTRATION (DAYS)

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260

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LUJ C-,

a.

B1O.D2 12.5 O-

mcg MCA

I 100 TIME AFTER ADMINISTRATION (DAYS)

200

TIME AFTER ADMINISTRATION (DAYS)

Figure 2-Survival of B13O.D2 female mice on the various diets after injection of 0, 12.5, or 125 ug methylcholanthrene in triactanoin subcutaneously. Groups of 25 mice.

DBA/2 mice given the vehicle, tractanoin, survived well unless they were fed the POLY diet (Figure 3). DBA/2 mice on all but the CHOW diet survived poorly if given 125 ,ug methylcholanthrene. Autopsies of the DBA/2 and the B 10.D2 mice revealed histologic evidence of pneumonia consistent with infection with Mycoplasma pulmonis. The mice housed in our "conventional" animal facility in the Animal Resource Center (ARC) usually are infected (as indi-

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ir V) z

L) a.,

D U)

z

L) Cdi

a-

TIME AFTER ADMINISTRATION (DAYS)

Figure 3-Survival of DBA/2 female mice on the various diets after injection of 0 or 125 ,ug methylcholanthrene. The symbols for the dietary groups are the same as in Figure 2.

cated by antibody titers) with Mycoplasma pulmonis, Sendai virus, and mouse hepatitis virus. The mice received from the Jackson Laboratory are specific pathogen-free, but they become infected in the ARC at the rate of about 10%/month. Several of the mice were sacrificed, and cultures of lung lavages revealed Mycoplasma pulmonis.

107

None of the experimental diets nor treatment with methylcholanthrene had stimulatory or inhibitory effects on IgM or IgG anti-SRBC agglutinin antibody responses in B1O.D2 or DBA/2 mice (Table 1). The "resting" NK activity of all of the tested 11month-old B 1 0.D2 and DBA/2 mice was low and was boosted moderately well by pl: pC (data not shown). None of the diets had a consistent inhibitory effect. NK and NC activities, as measured in vivo by lung clearance of radiolabeled YAC- 1 and WEHI- 164.1 cells, were not affected by diet (Table 2). B 10.D2 mice did clear WEHI-164.1 cells more efficiently than DBA/2 mice. DBA/2 mice on the MONO diet appeared to clear WEHI- 164. 1 cells relatively poorly. When tested for cutaneous sensitivity to trinitrochlorobenzene, all B10.D2 mice on the POLY diet showed a consistent pattern of immunosuppression, whether or not they were given methylcholanthrene (Figure 4). It should be noted that survival was good in the mice given the vehicle or only 12.5 jug methylcholanthrene. Healthy-appearing mice could be immunosuppressed, but even in these "healthy" mice, Mycoplasma pulmonis infection was common. Like the B1O.D2 mice, both groups of DBA/2 mice on the POLY diet developed poor cutaneous sensitivity responses to trinitrochlorobenzene (Figure 5). The DBA/2 mice on the MONO and SAT diets were also significantly suppressed (P< 0.05 versus CHOW diet), but to a much less degree than on the POLY diet. Again, note that survival of DBA/2 mice was poor when they were injected with the carcinogen and good in controls receiving vehicle, with the exception of mice on the POLY diet.

Studies of Male C3B6F1 Mice The C3B6F1 mice had excellent survival (95%) for the first 4 months and were clinically free from infec-

Table 1 -Antibody Responses of Mice on Diets and Given Injections of Methylcholanthrene (MC)* IgG titers (peak)

IgM titers (peak) Diet

No MC

12.5 jug MC

125 ,ug MC

No MC

12.5 ,ug MC

125 ug MC

38.0 10.0 25.0 15.0 13.1

8.6 15.0 10.9 5.5 33.0

13.9 19.0 20.5 10.5 7.9

9.9 8.0 2.9 2.9 4.0

13.2 4.9 4.0 8.0 3.2

11.6 23.9 3.4 3.4 6.6

14.0 6.0 10.5 15.5 3.4

-

36.0 91.9 13.9 13.5 6.5

8.0 9.4 6.6 4.6 4.6

-

8.0 8.0 8.0 6.4 2.9

1. B1 O.D2 Mice

CHOW LoFat POLY MONO SAT 11. DBA/2 Mice CHOW LoFat POLY MONO SAT

-

*Mice were immunized with 5 X 10. SRBCs intrapentoneally, and serum was obtained on Days 5, 10, and 18. The serum was treated or not with 2ME. The peak IgM titers were detected at Day 5, and the peak IgG (2ME-resistant) titers were detected on Day 18.

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Table 2 -Lung Clearance of Tumor Cells by Mice on Diets and Given Injections of Methylcholanthrene (MC)* WEHI-164.1 cells YAC-1 cells 12.5 jug MC No MC 125 ,ug MC 12.5 ,g MC No MC Diet l. B10.D2 Mice CHOW LoFat POLY MONO SAT II. DBA/2 Mice CHOW LoFat POLY

MONO SAT

125 jug MC

4.0 2.0 3.4 3.0 3.4

2.8 3.0 2.3 6.7t 4.6

4.2 4.2 3.9 2.4 2.3

11.1 6.5 7.6 8.6 8.3

5.4 8.0 4.9 10.7t 10.4t

8.4 10.1 7.3 6.6 8.3

6.3 4.0 6.2

-

16.9

38.4 42.5

-

-

10.3t

-

-

7.4

-

7.8 8.5 8.2 10.0 8.6

34.8 22.5

-

7.1 10.2

35.6t 18.1

-

22.5

*Mice were infused with 5 X 105 5'Cr-labeled YAC-1 cells and 5 X 105 1251UdR-labeled WEHI-1 64.1 cells intravenously. The lungs were removed 4 hours later. Values are expressed as geometric mean percent retention of infused cells in the lungs. tGeometric mean values significantly greater (P < 0.05) than those of the CHOW group.

tion, even though they were housed in the conventional quarters. The fatty acid contents of the mesenteric fat pads reflected the diets quite well (Table 3) and the body weights indicated that the diets were well consumed. These mice were tested for cutaneous sensitivity to TNCB 4 months after receiving the experimental diets. The data in Table 4 indicate that none of the

ABIO.D2 Mice Vehicle , , , , . . , ,., , , , , ...

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B10. D2 Mice 125 0ucg MC how CM LoFat

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Ear Thickness (xO.001 in.) 24h Figure 4-Cutaneous sensitivity to TNCB in diets and given vehicle (A), 12.5

*Significant

pg9

suppression (P < 0.05),

B1 0.D2 female mice on various pug methylcholanthrene (C).

(B), or 125

compared with CHOW controls.

20 25 15 10 5 Ear Thickness (x 0.001 in.) 24h

Figure 5-Cutaneous sensitivity to TNCB in DBA/2 female mice on various diets and given vehicle (A) or 1 25,ug methylcholanthrene (B). *See Figure 1.

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Table 3-Fatty Acid Contents of the Fat Pads of C3B6F1 Mice on the Various Diets for 7 Months and Mean ± SEM Body Weights at 6 and 11 months* Fatty acids C12 C14 C16 C16:1 C18 C18:1 C18:2 C18: 2 cis/trans C18:3 C20:3 w-6 C20:4 w-6 C? C20:3 w-9 C20:5 w-3 C22:6 w-3 Body weights(g) At 6 months At 11 months

CHOW 0 0.8 18.4

FISH

MONO

1.0 2.5 35.8 1.0 3.7 31.1 15.8 0.4 2.7 1.2 1.4 0 0

7.4

1.6 46.2 20.9 0.3 1.0 1.6 0 0 0 0.4 0

1.4 2.4

30.9 ± 2.7 34.9 ±1.8

32.7 ± 2.9 43.1t ± 1.9

POLY

Poly +ASA

SAT

0 0 8.8 0 0 74.0 11.8 0.8 1.5 0.3 1.2 0 0 0.4 0.2

0.3 0 12.8 0 0 19.3 64.7 0 0.8 0 0.6 0.5 0 0.5 0

0.4 0 17.6 0 0 23.5 55.6 0 0.9 0 0.5 0.8 0 0.5 0

0.3 0 13.2 7.0 64.4 11.1 0 0 1.3 0 0 0 0.9 0 0

45.7t ± 2.9 49.8t ± 0.8

40.4t ± 4.5 45.8t ± 2.2

42.8t ± 7.9 51.1t ± 1.0

47.9t ± 4.1 51.9t ± 1.0

*Intraabdominal mesenteric fat pads were removed and frozen immediately. Fatty acid content as a percentage of acids of 12 carbons or more was determined by gas chromatography. t Mean values significantly greater (P < 0.05) than those of CHOW controls.

diets were immunosuppressive. Mice were given injections of ethylnitrosourea (ENU) or vehicle intratracheally and were tested 1 month later. The mice given the vehicle were not immunosuppressed. The mice given ENU and receiving the MONO, POLY, or POLY + ASA diet had significantly depressed cutaneous sensitivity responses to TNCB (Table 4). Mice on the CHOW, FISH, or SAT diet were not suppressed. Survival at this point was excellent. A few mice that had received ENU were wheezing. Their lung lavage grew Mycoplasma pulmonis. The mice injected with ENU were sacrified 13 weeks later. The lungs contained histologic foci of Sendai virus pneumonitis and/or alveolar adenomas, which are difficult to distinguish. Mycoplasma pneumonitis, which contains lymphoid cell infiltrates predominantly, was detected to a lesser degree. The frequencies of lung lesions

(pneumonitis or adenommas) in the mice on the various diets are presented in Table 4. Mice on the FISH and SAT diets had the lowest incidences of lung lesions.

The preceding data suggested that the immunosuppression was induced in susceptible mice after carcinogen injection and may have been caused, in part, by infection. Therefore, C3B6F1 mice on the various diets were deliberately infected with Mycoplasma pulmonis intratracheally. These mice were tested for delayed-type hypersensitivity responsiveness to SRBCs and to H-2 allogeneic spleen cells. Only the mice on the POLY diet were significantly suppressed (Table 5). Aspirin in the drinking water prevented severe suppression of delayed-type hypersensitivity. The mitogenic responses to Con A and to LPS were vigorous by spleen cells from mice on the various diets

Table 4- Cutaneous Sensitivity Reactions to Trinitrochlorobenzene (TNCB) by C3B6F1 Mice Before and After Injection of Ethyinitrosourea (ENU) or Vehicle Intrattracheally*

Dietary group CHOW FISH MONO POLY POLY+ ASA SAT

Prior to ENU 7.3 ± 0.4 9.7 ± 0.8 7.8 ± 0.1 7.2 ± 0.3 9.0 ± 0.7 11.2 ± 0.5

Mean ± SEM increase in ear thickness at 24 hours (1 0-3 in) After ENU's vehicle After ENU 7.3 ± 0.3 9.9 ± 0.7 7.1 ± 0.3 8.8 ± 0.7 7.9 ± 0.3 9.3 ± 0.6

6.1 ± 0.6 7.7 ± 0.7 3.6 ± 0.2t 2.8 ± 0.7t 2.9 ± 0.3t 5.0 ± 0.3

Frequency of

lung lesionst 8/25 2/26 10/28 11/17 9/27 4/22

*Mice were sensitized to TNCB 8 weeks before the instillation of 6004ug ENU (or the vehicle, RPMI 1640 medium) intratracheally or were sensitized 4 weeks later. The mice were challenged 5 days after sensitization. tMean value significantly less than that of CHOW controls P