Expression of functions by normal sheep alveolar macrophages and their alteration by interaction with Mycoplasma ovipneumoniae

veterinary microbiology EUEVIER

Veterinary Microbiology 58 (1997) 31-43

Expression of functions by normal sheep alveolar macrophages and their alteration by interaction with Mycoplasma ovipneumoniae Mamadou Niang a31,Ricardo F. Rosenbusch b,*, Jose Lopez-Virella a, Merlin L. Kaeberle a ’ Deportment of Microbiology, Immunology and Prerentice Medicine, College of Veterinary Medicine, Iowa State University, AmesJA 5001 I, USA h Veterinay Medical Resenrch Institute, College of Veterinan’ Medicine, Iowu State Unic,ersify. Ames. IA 5001 I. USA

Received 13 March 1997: accepted 14 May 1997

Abstract Normal sheep alveolar macrophages collected by bronchial lavage were exposed to live or heat-killed Mycoplasma orlipneumoniae organisms, and their capability to ingest Staphvlococcus aureus and to elicit antibody-dependent cellular cytotoxicity against sensitized chicken red blood cells was tested. Controls consisted of non-infected macrophages in Ml99 medium. In addition, the effect of M. ovipneumoniae on expression of surface molecules on these sheep alveolar macrophages was determined. The percentage of S. aureus ingested by nontreated sheep alveolar macrophages was significantly higher than that of infected macrophages. Live mycoplasmas were more effective in suppressing the ingestion of S. aureus by these macrophages than killed mycoplasmas. Both live and killed mycoplasmas suppressed the cytolytic effect of the sheep alveolar macrophages to a similar degree. About 78% and 4.5% of the normal sheep alveolar macrophages had IgG and complement receptors, respectively. Infection of these macrophages with M. ocipneumoniae decreased significantly the expression of IgG receptors but had no effects on complement receptors. There were substantial increases in the expression of both MHC class I and class II by the mycoplasma-induced macrophages as compared with unstimulated macrophages. Live mycoplasmas were more effective in inducing expression of both classes than killed mycoplasmas. The results, taken together, suggest that M. octipneumonicle induced alterations in

_ Corresponding author. Tel.: + I-515-294-6170: ’ Present address: Central Veterinary Laboratory.

fax: + 1-515-294-1401. B.P. 2295, Bamako. Mali, West Africa.

0378-I 135/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SO378-1 135(97)00141-7

macrophage activities and this may be a contributing factor in the pathogenesis of respiratory disease induced by the organism. 0 1997 Elsevier hence B.V.

1. Introduction Alveolar macrophages represent a major component of pulmonary defense mechanisms against infectious agents. Alteration of their functional activities may have adverse consequences. Previous in vitro studies have shown that, in the absence of specific antibodies. several mycoplasma species not only can survive in cultures of phagocytic cells but can also interfere with ingestion and killing of a second bacterial target (Jones and Hirsch, 197 1; Simberkoff and Elbach, 197 1: Bredt, 1975; Powell and Clyde, 1975: Thomsen and Heron, 1979; Howard and Taylor. 1983; Almeida et al.. 19921. Also, mycoplasmas are capable of stimulating macrophages to release cytokines and other inflammatory mediators, and to express high levels of MHC molecules (Chelmonska-Soyta et al., 1994; Stuart et al., 1989, 1990). These mycoplasma-induced alterations of macrophage activities may contribute to disease pathogenesis by fdcilitating establishment of a secondary bacterial infection (Almeida et al., 1992) or bl induction of autoimmune antibodies and cell injury (Stuart et al., 1990). Although h~.vcopla.smu or~ipneumoniac is the most common mycoplasma isolated from ovine iungs (Davies, 1985; Khan, 1993), its role as a primary or secondary pathogen in the ovine respiratory tract is still not clearly defined. M. o~~pnrutnoni~~~ has been reported to colonize the surface of the sheep alveolar macrophages without being phagocytosed (Al-Kaisi and Alley, 1983). However, this study did not investigate the effects of the organism on the ability of these sheep alveolar macrophages to cause phagocytosis of a second target. Moreover. little is known about the in vitro culture characteristics (e.g., phagocytic activity. immune receptors) of sheep alveolar macrophages. The purpose of the present study was to characterize the sheep alveolar macrophages relative to their functional activity and the presence of immune receptors. Experiments were also performed to determine the effects of M. or,~~,lzeilrrrorziac, ijointed from iambs with a respiratory disease, on these sheep alveolar macrophage functional activities.

2. Materials

and methods

2.1. Macrophage

c~ollcction

Alveolar macrophages JAM) were collected by bronchial lavage of lungs obtained from freshly slaughtered lambs and healthy adult sheep that were sacrificed hy inrra 350 mp pentobarbi!~,r!e,,‘rll’; venous injection of Beuthanasia-D Special containing (Schering-Plough Animal Health, USA), 10 ml/animal. Animals were free of mycoplasmas as attested by bacterial culture of tracheal swabs taken deep into rhe trachea iu!+l

before the lavage. The trachea was immediately exposed and tied below the larynx with a piece of cord to avoid any influx of blood. The lungs were then carefully removed and two consecutive bronchoalveolar lavages were performed by infusing 500 ml of sterilt: warm phosphate buffered saline solution (Dulbecco’s PBS. without calcium chloride a:~1 magnesium chloride, pH 7.4) (Sigma Chemical, USA) containing 10 LJ heparin/s-ai (Gibco. Grand Island Biological, USA), 100 IU penicillin/ml (Gibco), 100 ~clg strcptt). mycin sulfate/ml (Gibco). 50 pg Gentamicin/ml (Sigma), and 0.125 pg fungizonc,,/ rlli (Gibco) into the lungs through the trachea. The lungs were gently massaged and this lavage fluid was poured into a sterile Erlenmeyer flask. The lava&e fluid was filtered through a double layer of cheesecloth into a sterile flask. Cells present in the Isvage fluid were washed twice in PBS by centrifugation (300 X g for 10 min) and the pellets were gently resuspended in Ml99 medium (with Earles’ salt solution, L-glutamine. sodium bicarbonate and Hepes buffer) (Gibco) supplemented with 10% fetal calf serum (FCS: Gibco) and antibiotics. The cell suspensions were depleted of red blood cellc. if necessary, by adding cold, sterile tris-buffered ammonium chloride (0.16 M NH,CI and 0.17 M tris-HCl, Ph 7.65) solution for 5 min at 37°C. centrifugation. washing and resuspension in Ml99 medium. A sample of the cell suspensions was streaked on a glass slide, stained with Diff-Quik stain (Baxter Scientific Products, IJSA), and a differenti,ti count was performed. The percentage of viability was determined by trypan blue exclusion. Total cell counts were made in a hemacytometer chamber (Hausser Scientific., IJSA) and the concentration of cells was adjusted as appropriate for each experiment. 2.2. Mwoplasma

preparation

The M. olipneumoniae isolate used for this study was cultured from a pneumonic sheep lung. The organism was identified as M. ovipneumouiae as previously described (Khan, 1993). A stock culture of the mycoplasma was prepared by growth in modified Friis broth medium (MFBM) (Khan, 1993). The number of organisms/ml was determined by the plate count method and aliquots were stored at - 70°C until used. Viability of the stock preparation was confirmed at the time of use in each experiment. Heat-killed mycoplasmas were prepared by incubating the organisms at 56°C in a water bath for 30 min. 2.3. Staphylococcus

aureus ingestiorl assay

Macrophage suspensions adjusted to 5 X lo6 ceils/ml were infected with M. olip~~eumcmiae to give a ratio of macrophages to mycoplasmas of 1: 1, 1: 10. 1: 100. 1: 1000. or 1:4000. Heat-killed M. ocipneumoniae were added only at a ratio of 1000 organisms per macrophage. Controls consisted of macrophages in MFBM or in Ml99 medium without added mycoplasmas. Since no major differences between the two control preparations were observed during the experiments, only Ml99 medium controls were utilized in reporting results. After an incubation period of 30, 60, 90, or 120 min at 37°C. the macrophages were washed twice with PBS by centrifugation (100 X 0” for 5 min) at 37°C their viability checked by trypan blue exclusion (greater than XO%t, resuspended in Ml99 medium and assayed for ““I-labeled Staph$ococcxs aureus

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M. Niang et al. / Veterinary Microbiology 58 ~1997131-43

ingestion as described previously (Roth and Kaeberle, 1981). The percentage aclreus ingested was calculated by using the following formula:

% ingestion=

2.4. Cytotoxicity

(CPM in test tube) - (CPM in background tube) (CPM . m standard tube) - (CPM in background tube)

of S.

X ]OO

assay

The antibody-dependent cell-mediated cytotoxicity (ADCC) assay was conducted with “Cr-labeled chicken red blood cells (CRBC) as target cells as described previously (Lukacs et al., 1985). Briefly, 100 ~1 of macrophage suspension in Ml99 medium at a concentration of 3.5 X IO6 cells/ml were placed in each well of a 96-well plate (Costar, USA). Then, 50 ~1 of diluted (1:4) bovine anti-CRBC antiserum (courtesy, Dr. J. Roth, Dept. MIPM, College of Vet. Med., Iowa State University, Ames, IA) and 100 ~1 of 51Cr-labeled CRBC were added to each well. After a 7 h incubation at 37°C in a 5% CO, humidified atmosphere, 5’ Cr release was measured by harvesting the supernatant onto cotton plugs (Skatron cell harvesting equipment, Skatron, Norway). The cotton plugs were transferred into tubes, placed in a gamma counter and counted for 2 min to determine the amount of radioactivity present. To determine if M. ovipneumoniae could interfere with the capacity of normal sheep AM to mediate ADCC, macrophage suspensions were incubated with live and heat-killed M. ovipneumoniae diluted in Ml99 medium (without antibiotics) at macrophage:mycoplasma ratios of 1: 10 and 1: 100. After 120 min, these macrophages were washed twice in PBS by centrifugation (100 X g for 5 mitt), their viability checked by trypan blue exclusion (greater than 80%) and assayed for cytotoxic activity. Each assay included a pair of standard wells which contained 150 ~1 of 1% Triton X-100 and 100 ~1 of ” Cr-CRBC but no macrophages or anti-CRBC serum. Also, a pair of (control) background wells which contained all reactants except macrophages were included. All samples were set up in duplicate. The percentage of j’ Cr-CRBC lysed was calculated by using the following formula: (CPM sample) - (CPM background) % lysis of target cells =

(CPM standard)

- (CPM background)

’ loo

2.5. Assay .for sur$ace receptors 2.5.1. Preparation of antibody-coated and complement-coated sheep red blood cells Sheep red blood cells (SRBC) were collected in an equal volume of Alsever’s solution and washed three times in normal saline solution by centrifugation at 500 X g for 20 min. The supematant was discarded and the packed cells were resuspended to a final 1% v/v concentration in PBS+ (Dulbecco’s PBS with calcium chloride and magnesium chloride; Ph 7.3) (Sigma). Erythrocyte-antibody complexes were prepared by mixing equal volumes of 1% SRBC suspensions with rabbit IgG or IgM anti-SRBC

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58 (1997) 31-43

35

serum (Cappel, Organon Teknika, USA) that were diluted in PBS+ at a subagglutinating titer (1:320). The mixtures were incubated for 30 min at 37°C and for an additional 30 min at 4°C. These complexes were designated as erythrocyte-IgG (EG) and erythrocyte-IgM (EM), respectively. For the preparation of erythrocyte-complement (EC) complexes, EM at a 1% concentration were washed once in PBS+. Freshly collected sheep serum (known to not agglutinate SRBC alone) was added at a rate of 100 pi/ml as a source of complement. The mixture was incubated for 30 min at 37°C. Unsensitized SRBC (E) were prepared by incubating washed SRBC in PBS+ in the absence of Ab or C for 30 min at 37°C and for an additional 30 min at 4°C. The various SRBC preparations were centrifuged at 300 X g for 5 min, and cells resuspended to a final concentration of 0.5% in Ml99 medium. These cells were used immediately in the rosetting assays. 2.5.2. Rosette formation Three ml of a suspension of macrophages (1 X 106/ml) were dispensed into a polystyrene 25 cm’ tissue culture flask (Coming Glass Works, USA) and incubated at 37°C in a 5% CO, humidified atmosphere to allow macrophages to adhere. After a 4 h incubation period, the cell monolayers were washed gently with warm sterile PBS to remove non-adherent cells. The number of adherent macrophages was estimated by using 0.2% trypsin (Difco Laboratories, USA) to release the cells in one flask. The number of cells was then counted in a hemacytometer. The flasks were reincubated overnight (about 10 h) in Ml99 medium containing 10% FCS and antibiotics. Macrophages in some flasks were infected with live A4. ouipneumoniae diluted in Ml99 medium (without antibiotics) to give a macrophage:mycoplasma ratio of approximately I:50 and incubated overnight. Observations indicated that the viability of macrophages was not affected by the 10-h infection with mycoplasmas. For the rosetting assay, adherent macrophages were washed once with PBS, and 2 ml of a SRBC preparation (EG, EM, EC, and E) were added to an individual flask (in duplicate). Incubation was allowed to proceed 45 min at 37°C in a 5% CO, humidified atmosphere. At the end of this time, the flasks were gently rinsed twice with PBS to remove unattached SRBC, and the macrophages were stained with Diff-Quik stain. The bottom of each flask was removed, and the cells observed through an oil immersion microscope at a 100 X magnification. A macrophage with at least 3 SRBC associated with its membrane was considered a rosette. A total number of 200 macrophages were counted randomly for each flask, and the percentage of rosette forming cells (RFC) was calculated as: Number of RFC % RFC=

Total number of counted cells( 200)

x 100

2.6. Assay for MHC molecules Macrophages a concentration were placed in the presence or

cultured in flasks were harvested and resuspended in Ml99 medium at of 1 X lo6 cells/ml. Two hundred ~1 of the macrophage suspensions each well of a 96-well, flat bottom sterile microtiter plate (Coming) in absence of live or heat killed preparations of M. ouipneumoniae to give

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a macrophage:mycoplasma ratio of about I : 100. After an incubation of I or 2 days, the medium was removed from the wells and macrophages were fixed with cold acetone for 5 min and washed three times (5 min each) with warn1 Hanks balanced salt solution without phenol red (HBSS) (Sigma) containing 5% FCS. All subsequent washes were performed in this manner, and all dilutions were made in the same HBSS. One hundred ~1 of mouse anti-bovine class I or class II monoclonal antibodies species cross-reactive (VMRD, USA), each diluted at 1: 100, were added to an individual well. After an incubation of 90 min at 37°C the plates were washed. and labeled with 100 ~1 of either fluorescein-conjugated (Kirkegaard & Perry Laboratories, USA) or R-phycoerythrin-conjugated (Sigma) goat anti-mouse antibody (IgG gamma-chain specific) diluted at 150 and 1: 11, respectively. After incubation and washing, the plates were read in a fluorometric microtiter plate reader (Cambridge Technology, USA) at appropriate excitation and emission filter wavelengths for each fluorochrome. Results were reported as fluorescent values. The specificity of the test system was detenined by inclusion of several background control wells in each plate. These consisted of wells with mycoplasmas or unstimulated macrophages, reacted with each of the secondary conjugated antibodies alone in the case of the macrophages, or with each of the monoclonal antibodies and the secondary conjugates in the case of the mycoplasmas. Fluorescent values of the background control wells were quite similar to those of empty wells in each plate demonstrating that nonspecific binding of the reagents was minimal. Of the two conjugates used, R-phycoerythrin appeared to be the most sensitive. Therefore results of the fluorescence measurements with the R-phycoerythrin conjugate were reported. 2.7. Statistical analysis Results differences

were analyzed by analysis of variance (ANOVA). The significance of between means was accepted when the P value was at least less than 0.05.

3. Results 3. I. E@ct of’ mycoplasmas

on S. aureus inge.stion by sheep AM

There was a non-linear dose-response in S. aureu,s ingestion when sheep AM were exposed to live M. wipneumnniae at ratios of 1: 1, 1: 10, 1: 100, l:lOOO, or I:4000 macrophage to mycoplasmas at all times tested (data not shown). The percentage of S. aureus ingested by nontreated sheep AM was significantly higher (P < 0.05) than that of preparations of AM pretreated with live or killed mycoplasmas at all times tested. Live mycoplasmas were significantly (P < 0.05) more effective in suppressing the ingestion of S. aureus by sheep AM than killed mycoplasmas (Fig. 1). 3.2. Effect of myoplasmas

on ADCC actir+t?, qf sheep AM

The percentage of “Cr released from CRBC by normal sheep AM (42%) was significantly higher (P < 0.05) than that for sheep AM treated with live or heat-killed

M. Niang et al. / Veterinay

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Microbiolop

58 (IYY71 31-43

31

n Untreated (controls) sheep AM aSheep

AM treated with HK mycoplasmas

n Shesp AM treated with live myccplasmas

90 60 Time (in minutes) Fig. 1. Effects of live, or heat-killed (HK) M. o~~ipneumoniue on .S. aweus ingestion by sheep AM at macrophage:mycoplasma ratio of 1: 1000. Controls consisted of untreated sheep AM in Ml99 medium. Values represent mean percentages (+SEM) of five separate experiments. Statistical differences are indicated as a = P < 0.05 from control values, and b = P < 0.05 for live vs. HK mycoplasmas.

M. ocipneumoniae (about 12% and 16%, respectively) (Fig. 2). There was no significant difference (P > 0.05) between live and heat-killed mycoplasmas in depressing sheep AM ADCC function. Similarly, increasing the ratio of M. ocipneumoniae (live and heat-killed) to sheep AM from IO:1 to 1OO:l did not significantly affect the ability of these AM to perform ADCC. 3.3. Effect qf m_vcoplasmas

011

rosette~formation

on sheep AM

Both non-infected sheep AM and those infected with live M. odpneumoniae failed to form rosettes with IgM-sensitized or with nonopsonized SRBC, while rosettes were observed with IgG-sensitized and C-sensitized SRBC (Fig. 3). About 78% and 45% of the normal sheep AM formed rosettes with IgG-sensitized or C-sensitized SRBC.

50

IllUntreated (control) sheep AM

qShefq~ AM

T

treated with live mycoplasmas

OSheep AM treated with HK mycoplasmas

Contrd

I:10

1:lOO

Ratio of macrophage to mycoplasmas Fig. 3. Effects of live, or heat killed (HK) M. ouipneumoniae on sheep AM cytotoxic activity at ratios of 1:10, or 1:100 macrophage to mycoplasmas. Values represent mean percentages ( f SEMI of three separate experiments. Statistical differences from control vlaues are indicated as; a = P < 0.05.

M. Niang et al. / Veterinary Microbiology 58 (1997) 31-43

38

100 -

E Untreated sheep AM 2 rY

sot

z

607

a

1 q Treated sheep AM

a, g

40

s i

f *o{ ~ OC

EM

E

EG

EC

Fig. 3. Percentages of sheep AM forming rosettes with various SRBC preparations. Sheep AM were either untreated or exposed to live M. ouipneumoniae. SRBC preparations were: E = nonsensitized, EM = sensitized with IgM, EG = sensitized with IgG, and EC = sensitized with complement. Values represent mean percentages (*SEM) of 10 separate experiments. The difference between a and b was significantly different (P < 0.05).

respectively. The mean percentage of infected sheep AM forming rosettes with IgGsensitized SRBC was 58%. This was significantly lower (P < 0.05) than that of non-infected sheep AM. C-sensitized SRBC formed rosettes with 49% of AM infected with M. ovipneumoniae which was similar to the value of non-infected AM. 3.4. Effect of mycoplasmas

on MHC molecule expression

on sheep AM

There was a measurable increase in the expression of both MHC class molecules when macrophages were stimulated by M. ovipneumoniae (Tables 1 and 2). Live organisms, at all times, were more effective in inducing expression of both classes than killed organisms. Live mycoplasma organisms induced an increase in both class I and

Table 1 Effects of M. ouipneumoniae (MO) on expression Dav

1

2

Treatment

Control MO-live MO-HK Control MO-live MO-HK

Fluorescence

of MHC class II on sheep alveolar macrophages

values

Exp. 1

Exp. 2

Exp. 3

129 2163 1419 358 643 388

634 1824 I148 288 493 361

176 2133 1409 506 719 486

Mean( f SD) 713.00+72.33 2040.00+ 1S7.66b.c 1325.33+ 153.65b 384.00+ 111.30 618.33&115.00 411.66+65.77

Macrophages were cultured alone (control), or in the presence of live or heat killed (HK) mycoplasma organisms. After 1 or 2 days incubation, macrophages were reacted with anti-MHC class II monoclonal antibodies followed by labeling with R-phycoerythrin conjugated second antibody. “Mean values (*SD) of the three separate experiments (Exp.). bSignificantly higher (P < 0.05) than the respective control value. ‘Significantly higher (P < 0.05) than the respective MO-HK value.

M. Niang et al./ Veterinary Microbiology Table 2 Effects of M. ouipneumoniae Day

1

2

Treatment

Control MO-live MO-HK Control MO-live MO-HK

(MO) on expression Fluorescence

58 CIYY7I 3i-43

39

of MHC class I on sheep alveolar macrophages

values

Exp. 1

Exp. 2

Exp. 3

2094 3912 2377 1324 1189 897

1126 3055 1945 959 942 721

2023 4046 2466 1197 1099 1079

Mean ( + SD)” 1941.66+ 195.22 3691.00+552.03b,C 2262.66 + 278.68 1160.OOf 185.29 1076.66 + 125.00 899.00 k 179.00

Macrophages were cultured alone (control), or in the presence of live or heat killed (HK) mycoplasma organisms. After 1 or 2 days incubation, macrophages were reacted with anti-class I monoclonal antibodies followed by labeling with R-phycoerythrin conjugated second antibody. ’ Mean values (k SD) of the three separate experiments (Exp.). b Significantly higher (P < 0.05) than the respective control value. ’ Significantly higher (P < 0.05) than the respective MO-HK value

class II molecules. That rise was more than a one-fold increase at Day I as compared with levels of expression on respective unstimulated macrophages. By Day 2, however, expression was decreased, in particular for class I, to a level below that of unstimulated controls. A separate experiment was performed to examine the proportion of normal macrophages expressing each of the MHC classes. Macrophages were cultured alone on Teflon-coated microscope slides (Gel-line, Newfield, NJ), and monolayers were subsequently reacted with the monoclonals and the fluorescein conjugate and examined under a fluorescence microscope. The number of fluorescent cells reacting with the anti-class I antibodies was far greater than the number reacting with the anti-class II antibodies (data not shown). Also, great variation in the intensity of the positively stained cells was noticed between cells reacted with each monoclonal.

4. Discussion The phagocytic effectiveness of phagocytic cells depends heavily on their membrane receptors which mediate both attachment and ingestion of foreign particles. Evidence is presented here to suggest that normal sheep AM express receptors for IgG and C but not for IgM. IgG receptors were present on about 78% of the normal sheep AM, while only 45% of these cells exhibited C receptors. This is quite similar to the IgG and C receptor percentages reported for equine (Dyer and Wes Leid, 19831, swine (Harmsen and Jeska, 1979), and guinea pig (Hunninghake and Fauci, 1977a) normal AM. However, in man and rabbits (Reynolds et al., 1975) the percentages of cells with C receptors reported were 93% and 80%, respectively. The absence of AM forming rosettes with IgM-sensitized SRBC found in the present study has been previously reported in swine (Harmsen and Jeska, 1979) and equine (Dyer and Wes Leid, 1983). Few reports have been concerned with surface receptors on AM from ruminants. McGuire and Babuik (1982) reported that almost 100% of bovine AM that had been cultured in vitro for at least 48 h

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expressed receptors for both IgG and C, but when examined earlier only 29.9% and 53% of these cells had IgG and C receptors, respectively. These authors suggested that the use of unfractionated rabbit antisera accounted for the low percentages seen in these early cultured cells, but as the cells matured in vitro, the expression and the number of cells displaying both Fc and C receptors increased. Fleit et al. (1986) used a fluorescence assay technique and reported that 62.7% of AM obtained from 5 day-old lambs had receptors for IgG, but they did not investigate the expression of C receptors. Our results indicate that 78% of normal AM obtained from adult sheep expressed IgG receptors. The technique employed and age-related differences may account for the difference between these two observations. The effect of M. ovipneumoniae on the expression of Fc and C receptors on normal sheep AM was investigated in the present study. Treatment of sheep AM with live mycoplasmas resulted in about a 20% decrease in sheep AM expressing IgG receptors, while the percentage of AM exhibiting C receptors remained almost constant, as compared with the normal sheep AM. The mechanisms involved and why C receptors were not affected remain undetermined. However, it is likely that this suppression of the IgG receptors by A4. ovipneumoniae can explain, in part, the impaired ability of these sheep AM to ingest S. aureus or to lyse 5’Cr-CRBC when pretreated with M. oLipneumoniae as demonstrated by the phagocytosis and cytotoxicity assays during the present study. Exposure of normal sheep AM to M. ovipneumoniae at low ratios of I- 10 mycoplasmas per macrophages decreased up to one-third the bacterial uptake by these phagocytic cells. Decreased activity was also observed when sheep AM were exposed to heat-killed M. ovipneumoniae but to a more limited degree as compared to live mycoplasmas. Also, a steady decline in S. aureus uptake by sheep AM was observed when these macrophages were pretreated with increasing concentrations of M. orjipneumoniae. This suggests a dose-related response. However, varying the time interval of sheep AM infection with M. oc!ipneumoniae did not affect the ingestion of S. aureus by sheep AM. An inhibitory effect in vitro of several mycoplasma species on the ability of phagocytic cells to phagocytose a second bacterial target has previously been reported (Simberkoff and Elbach, 1971; Thomsen and Heron, 1979; Howard and Taylor, 1983; Almeida et al., 1992). Similarly, certain microorganisms other than mycoplasmas have also been reported to interact with normal phagocytic cells in vitro, resulting in an impaired ability to phagocytose a second bacterial target (Hubbard et al., 1986; Roof and Kramer. 1989). The role of phagocytic cells in host-defence mechanisms depends also on their ability to lyse cells infected with various microbes through ADCC or complement-dependent cytotoxicity. Results of this experimentation indicated that normal sheep AM were quite effective in lysing 5’Cr-CRBC (in the presence of specific antibody) as target cells. When treated with both live and heat-killed M. ouipneumoniae, a significant loss of ability of these sheep AM to lyse the target cells was observed. These results clearly differ from those of Loewenstein and Gallily (1984), who reported that both live and heat-killed M. orale stimulated macrophage-mediated cytolysis of mouse tumor cells. Increased ability of murine macrophages treated with M. capricolum membranes to lyse tumor target cells has also been reported @her et al., 1990). Species variation and nature of the target cells may account for these differences.

Little is known about the mechanisms that allow mycoplasmas to suppress phagocytic cell functions. However, evidence exists that the capsular material may inhibit the ability of phagocytic cells to ingest and kill a second bacterial agent. Almeida et al. (1992) demonstrated that encapsulated M. dispcrr or its purified capsule inhibited the phagocytosis of S. aureus or Serratia marcescens by bovine AM. Our recent studies have shown that M. or?pneumoniae organisms are also encapsulated (Niang et al., 19941. Whether or not the capsule of the organism has something to do with these inhibitory effects on sheep AM functions is not known, but analogy with M. dispnr capsule can be implied. Also, the ability of mycoplasmas to inhibit phagocytosis may result from their ability to bind firmly to the cell membranes of these phagocytes. Electron microscopic studies by Al-Kaisi and Alley (1983) showed that, in the absence of specific antibodies. M. o[~ipneumonine remained attached to the surface of sheep AM without being phagocytosed. This could mask or prevent stimulation of appropriate receptors (Howard and Taylor, 198.5). The finding in the present study that M. oL:ipneumoniae decreases the number of IgG receptors on sheep AM may be correlated, at least in part, to the impaired ability of these macrophages to ingest S. nureus or to lyse CRBC as target cells. Several conditions have been shown to modulate Fc and C receptor expression. Harmsen and Jeska (1979) reported a 45% increase in C receptor expression in pigs experimentally infected with Toxoplasma gondii, while the percentage of AM exhibiting IgG receptors remained constant compared to the non-infected control pigs. However, they concluded that this augmentation of C receptors did not independently enhance the ability of these pig AM to endocytose SRBC. These results differ from those that we have found. Species variations or conditions of experimentation may explain these differences. Caruso and Ross (1990) reported that the percentage of AM phagocytosing opsonized sheep RBC was not affected by in vivo infection of pigs with M. h_vopneumoniae alone. but was suppressed by a combined infection with M. hyopneumoniae and Actinohacil-, lus plel(rt,pneumoniae. Other studies have shown that prolonged exposure of guinea pigs to corticosteroids in vivo can cause alterations in membrane Fc receptor function of AM (Hunninghake and Fauci. 1977b) and the loss of these Fc receptors caused a marked decrease in cytotoxic effector function of these macrophages (Hunninghake and Fauci, 1977~). Similarly. administration of glucocorticoids decreased the number of IgG receptors on phagocytic cells from man (Parrillo and Fauci, 1979). While our data seem consistent with these findings, the manner in which this inhibitory effect is exerted remains to be determined. Perhaps there is release of certain substances that might alter the phagocytic cell surface membrane or metabolism. However, even with such an hypothesis. why the C receptors were not also affected is puzzling. The present study indicated that normal sheep AM express both MHC class 1 and class II molecules on their surface but level of class I expression was far greater than that of class II as judged by immunofluorescence staining. Also, the extent of expression of both classes vary between individual cells. Live or heat-killed M. oripneumonirre induced increased expression of both MHC classes on the macrophages. This finding contrasts with the inhibitory activity of the organism on other macrophage characteristics. However. these differential effects may be characteristic of mycoplasmas in

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general since M. hominis, M. arthritidis and M. pulmonis suppress the phagocytic function of phagocytes (Simberkoff and Elbach, 197 1; Thomsen and Heron, 1979; Howard and Taylor, 1983) and upregulate MHC molecule expression on macrophages (Stuart et al., 1989). This induced MHC molecule expression on macrophages may help lead to the development of autoimmune disorders (Stuart et al., 1990). Results of the present study suggest that M. ouipneumoniae can induce alterations in sheep AM activities. These in vitro inhibitory effects of M. ovipneumoniae on the ability of sheep AM to ingest a second bacterial agent, to lyse a target cell, and the diminished expression of IgG receptors on macrophages combine with increased MHC antigen expression on these macrophoges, define a potential role that this organism may play in sheep respiratory disease.

Acknowledgements The authors are grateful to Dagmar E. Frank and Anna Rovid (Department of Microbiology Immunology and Preventive Medicine, College of Veterinary Medicine, Iowa State University) for technical assistance, to Kevin Flaming (Department of Microbiology Immunology and Preventive Medicine, Iowa State University) and LieLing Wu (Department of Statistics, College of Liberal Arts and Sciences, Iowa State University) for statistical assistance. This work was supported in part by grants from the USDA and the Iowa Livestock Health Advisory Council.

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