Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production

Veterinary Immunology and lmmunopathology, 31 (1992) 77-94

77

Elsevier Science Publishers B.V., Amsterdam

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production Andrew D. Nash ~, Garry J. Barcham, Arna E. Andrews and Malcolm R.

Brandon Centrefor Animal Biotechnology, Department of Veterinary Science, The University of Melbourne, Parkville, 3052, Vic.,Australia (Accepted 28 February 1991 )

ABSTRACT Nash, A.D., Barcham, G.J., Andrews, A.E. and Brandon, M.R., 1992. Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production. Vet. Immunol. Immunopathol., 31: 77-94. Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFNT) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFNy increased surface expression of MHC class I and class II (DRtx, DPct, and DQa) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN7 specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN~ required for increased class II expression was 0.1 U ml- ~and for increased class I expression, 1 U ml- 1. Northern blot analysis indicated that rIFN~,mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human eDNA probes we failed to detect mRNA encoding the cytokines IL-la, IL-1fl, and TNFt~ in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNFct mRNA was induced to maximal levels within 1 h, declining thereafter. IL-lct mRNA was detected at l h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-lfl mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-i induced detectable levels of TNFct mRNA while levels as low as 10 pg ml-l induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.

INTRODUCTION

Cytokines play a key role in the regulation of macrophage functional capacity during antigen specific and non-specific immune responses, firstly as mo1~Author to whom correspondence should be addressed.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

78

A.D. NASH ETAL.

ecules that directly influence the functional status of macrophages (Cao et al., 1989) and, secondly, as the effector molecules of activated macrophages (Oppenheim et al., 1986; Harrison and Campbell, 1988 ). Although sheep are important as experimental animals for studies of lymphocyte recirculation (Mackay et al., 1988) and immunity to infectious disease (Meeusen et al., 1990) there are no reports describing cytokine mediated regulation of ovine macrophage functional capacity. This report examines the phenotypic changes induced by recombinant interferon gamma (rlFNT) on ovine alveolar macrophages and the production of IL-1 and TNFo~ by alveolar macrophages activated with lipopolysaccharide (LPS). In human and murine models IFN-), (Kelley et al., 1984; Wong et al., 1984), as well as other cytokines (Fertsch et al., 1984; Chang and Lee, 1986; Stuart et al., 1988 ), have been demonstrated to be potent regulators of MHC antigen expression on macrophages. As the clonotypic T cell receptor recognises antigen in the context of MHC class I or class II molecules (Townsend and Bodmer, 1989 ) this regulation has important implications for both initiation and development of immune responses. Synthesis of IL-1 (two molecules, I L - l a and IL-lfl) and TNFo~ by macrophages can also result in profound effects on the immune response, as these two cytokines are important mediators of the inflammatory response inducing secretion of acute phase proteins, stimulating release ofprostaglandin E2 and proteolytic enzymes, and enhancing cellular infiltration through chemotaxis and mitogenicity (Mawatari et al., 1982; Kampschmidt, 1984; Dayer et al., 1985 ). IL-1 also plays an important role in antigen specific immune responses acting as an early signal in T cell activation and participating in the cytokine network that regulates B cell differentiation (Oppenheim et al., 1986). The results presented in this report characterise the effects ofrlFN7 on MHC class I and class II molecules at both the level of transcription and cell surface expression. Induction of IL- 1 and T N F a by LPS was examined at the level of transcription and, for IL- 1 secretion of bioactive protein. MATERIALS AND METHODS

Isolation and culture of alveolar macrophages Lungs and trachea were removed intact from 1- to 2-year-old Merino ewes housed at the Department of Veterinary Science, University of Melbourne. Lungs were then flushed via the trachea with 400-800 ml of sterile phosphate buffered saline (PBS). Cells were recovered from lung washings by centrifugation, and washed twice in RPMI-1640 supplemented with 10% v/v fetal calf serum, 2 mM glutamine, 100 U ml-1 penicillin and 0.1 mg m l - l streptomycin (RF 10 ). Cell populations were cultured in 80 cm 2 tissue culture flasks in RF10 at 37°C with 5% CO2 in air. After 1 h cell monolayers were gently

OVINE ALVEOLAR MACROPHAGES

79

flushed with two changes of RF 10 to remove any non-adherent cells. Nonspecific esterase staining and surface antigen expression indicated that in all cases more than 98% of adherent cells were macrophages. Where indicated macrophages were stimulated with either bacterial LPS (Sigma Chemical Co) or bovine rlFN7 (gift of Dr. P. Wood, CSIRO Division of Animal Health, Melbourne, sp. act. 2.2 × 106 U mg -1 ) at the appropriate concentration (see results). Macrophages were recovered for flow cytometry and RNA preparation by trypsinisation. For all experiments described, a minimum of three and maximum of five donors were used with representative data presented.

Monoclonal antibodies The monoclonal antibodies (mAbs) used in this study were directed against the ovine cell surface antigens CD4, CD8, T19, CD5, CD45, CD45R, and MHC class I and class II molecules and have been previously described ( Mackay et al., 1986, 1987; Puri et al., 1987 ). mAbs re active with MHC class II molecules included 49.1 (all class II molecules), 42.20 ( D R a specific), 38.27 ( D Q a specific) and 28.1 ( D P a specific). ILA-24 is a mAb specific for bovine myeloid cells that cross-reacts with ovine myeloid cells and was obtained from Dr. J. Ellis, Wyoming State Veterinary Laboratory (Ellis et al., 1987 ). mAbs against bovine rlFN7 (IFN2 and IFN9 ) were a gift from Dr. P. Wood and their characterisation including ability to neutralise IFN bioactivity has been previously described (Wood et al., 1990).

Immunofluorescence staining andflow cytometry Single colour immunofluorescence staining of macrophages was performed as described previously (Mackay et al., 1986). Briefly, 1-2 × 106 cells were incubated with 50/zl ofmAb as undiluted supernatant fluid (SNF) in microtitre trays. After washing, cells were reacted with 50 #l of a 1 : 30 dilution of FITC-conjugated F ( a b ) 2 fragments of sheep anti-mouse immunoglobulin (Silenus, Melbourne). All reactions were performed at 4 oC. All samples were fixed with 1% formaldehyde, 2% glucose in PBS and analysed on a fluorescence activated cell analyser (FACScan, Becton Dickinson, Ca.).

Bioassays The NOB- 1/CTLL assay for determination of IL- 1 has been previously described (Gearing et al., 1987 ). Briefy, NOB-1 cells were washed three times in RFI0, resuspended at 2 × 10 6 m l - 1 and 0.1 ml added to 'V' bottom microtitre plates containing 0.1 ml of appropriately diluted test SNF. After incubation for 24 h at 37°C, plates were centrifuged at 1000 rev min -1 and 50#1 of SNF transferred to a replicate fiat bottom microtitre plate together with 50

80

A.D. NASH ET A L

/~1 containing 5 X 10 3 CTLL cells. For determination of proliferation, CTLL were pulsed at 20 h with 3H-thymidine (Amersham) and harvested 2 h later. All test samples were assayed in duplicate with standard errors, which are not shown, always less than 10%. The actinomycin D treated L-cell or WEHI- 164 assay for measuring TNFo~ activity was performed basically as previously described (Renz et al., 1988 ).

Histochemistry Non-specific esterase staining was performed on alveolar macrophages using the methods described by Koski et al. ( 1976 ).

DNA probes cDNA probes encoding human IL-1 c~ and IL-lfl and human T N F a were generously provided by Immunex Research and Development Corporation (Seattle, WA) and, Genentech Inc. (South San Francisco, CA) respectively. The ovine MHC class II probe was a genomic clone encoding an OLA DRA gene (Scott et al., 1987 ). The mouse MHC class I probe was a 2.4 kb BamH 1 fragment containing exons 4, 5, 6, 7 and 8 of a H-2K b gene (Weiss et al., 1983 ) and was obtained from Dr. J. McClusky, Department of Immunology and Pathology,Monash University. cDNA probes encodingfl-tubulin (Cleveland et al., 1980) or GAPDH (Piechaczyk et al., 1984) were used to indicate relative levels of RNA. 32p-labelled probes were generated by random priming essentially as previously described (Sambrook et al., 1989 ).

RNA isolation and Northern blot analysis For preparation of RNA, fresh or cultured macrophages were washed twice in PBS. Total RNA was then prepared from the cell pellet by acid phenolguanidine thiocyanate extraction (Chomczynski and Sacchi, 1987). RNA samples were fractionated by electrophoresis through denaturing formaldehyde-agarose gels and transferred to Hybond-N nylon membranes (Amersham) as previously described (Sambrook et al., 1989). Following overnight transfer RNA was fixed to membranes by UV cross-linking and hybridised to appropriate 32p-labelled probes (Sambrook et al., 1989). When using ovine probes, blots were washed under conditions of high stringency (0.5 x SSC, 60°C) following hybridisation. For cross-species probes low stringency ( 1 X SSC, 50 ° C) conditions were employed. The 28S and 18S ribosomal bands were used as markers.

OVINE ALVEOLAR MACROPHAGES

81

RESULTS

The cell surfacephenotype of ovine alveolar rnacrophages Alveolar macrophages were recovered from lung washings and subjected to immunofluorescent staining with a panel of mAbs specific for ovine leukocyte cell surface antigens. Surface antigen expression on macrophages did not vary between the five sheep tested with cells not staining for the T cell surface antigens CD4, CD8, CD5, and T19 as well as CD45R. All macrophages expressed CD45 and all were MHC class I positive. Greater than 90°/0 of macrophages stained positively for the' MHC class II molecules DRop, DPa, and DQa. A similar pattern of staining was observed with mAb 49.1 which reacts with all ovine class II molecules (Fig. 1A). All alveolar macrophages recovered were esterase positive.

A

:::'::: e

u I I

. .... ;I,

'"i 2

.....

.......

;12

....

.......

%

im q I

....

';2,

....

LOG FLUOR(SCEHC[ IHTEHSITY

Fig. 1. FACS analysis of the regulation of alveolar macrophage MHC antigen expression by rIFN~,. Alveolar macrophages cultured for 48 h in RF 10 ( . . . ) or RF 10 supplemented with 10 U m l - 1 of ~ I F N (....) were stained with mAb specific for MHC class II (49.1, A) or class I (B) molecules, rIFN~ treated populations were also stained with a negative control SNF ( - - ) . Histogram C shows MHC class II (mAb 49.1 ) staining of macrophages incubated in RF10 ( - ) , RF 10 supplemented with 10 U m l - t rIFN~ ( . . . ) , or RF 10 supplemented with 10 U m l - t rlFN~ and a 1 : 2000 dilution of rIFN~, specific mAbs. (....).

8,2

A.D.NASHETAL.

The effects of rlFN7 on macrophage surface antigen expression In the following experiments the ability of bovine rlFNy to modulate surface antigen expression on macrophages from five sheep was examined. Results of a representative experiment are presented. Alveolar macrophages were cultured in RF10 and in R F I 0 supplemented with 10 U ml-1 o f r l F N 7 or I0 /tg m l - t of LPS. Cells were recovered at 24 and 48 h, stained with mAbs against MHC class I and II antigens, CD45 and ILA-24. Reactivity was assessed by flow cytometry. Neither rlFNy nor LPS influenced the level of expression of CD45 or ILA-24 antigens after 24 h or 48 h in culture (results not shown). In contrast, rlFNy did upregulate MHC class I and II expression with the effect being apparent at 24 h and maintained for the next 24 h. Figure 1A shows that at 48 h, the presence o f r l F N y had decreased the heterogeneity in the level of class II expression observed and increased the mean level of fluorescence intensity compared with incubation in m e d i u m alone. The results presented in Fig. 1A were obtained using the mAb 49.1 which reacts with all class II molecules. Identical results were obtained when cells were stained with mAbs specific for DRc~, DQc~ or D P a class II molecules (not shown). Increase in the level of fluorescent staining observed with class I specific mAb, although less dramatic than that observed for class II, was readily apparent (Fig. 1B). Culture with 10/tg m l - 1 of LPS did not affect class I or class II antigen expression (not shown). To verify that the upregulation in MHC antigen expression was due to rlFNT, we made use of two mAbs specific for bovine rlFNy. Macrophages were incubated for 48 h in RF 10 alone, RF 10 supplemented with 10 U m l of rlFN7 and R F I 0 supplemented with 10 U m1-1 of rlFNy plus a 1:2000 dilution of ascites of the rlFN~, specific mAbs INF2 and IFN9. Results shown in Fig. 1C indicate that there was significant upregulation of class II expression induced by rlFNy and that this effect was totally abrogated by the presence of the IFNy specific mAbs. Shown in Fig. 2 is a dose response curve for the induction of MHC class I and II molecules by rlFNT. The results represent determinations using macrophages from a single sheep however a similar dose response has been determined using two other donors. Macrophages were incubated for 48 h in RF10 alone, or in 10-fold dilutions of rlFNy ranging from 100 to 0.001 U m1-1. Macrophages were recovered and stained with the class II specific mAbs 49.1 and 38.27, a class I specific mAb and mAb ILA-24 and mean fluorescence intensity determined by FACS analysis. As expected the level of ILA-24 surface expression was not altered over the entire dose range of rlFNy tested. The intensity of staining observed with both class II specific mAbs decreased markedly when the level of rlFN7 was reduced below 10 U ml-~ until at 0.01 U ml-~ only control levels of class II expression were observed. The dose

OVINE ALVEOLAR MACROPHAGES

83

1,~0

1200 e

,"r

600'

i

| 300,

O

o

u

•

u

lOO i0

l

0.!

i

l

.01.00l .OOMMED

Conc. glFN (units/ml)

Fig. 2. Dose response for the induction of MHC class I and II expression on alveolar macrophages by rIFN~. Alveolar macrophages cultured for 48 h in RF10 (isolated symbols) or RF10 supplemented with dFN7 (joined symbols) were stained with the MHC class II specific mAbs 49.1 (~q-[]) and 38.27 ( 0 - 0 ) , an MHC class I mAb ( i - n ) , and mAb ILA-24 ( ~ - ~ ) and reactivity assessed by FACS analysis.

response varied slightly for class I induction with control levels of expression observed for levels of rIFN7 below 0.1 U m l -

Northern blot analysis of MHC antigen regulation by rlFN7 FACS analysis of macrophage surface antigen expression indicated that incubation with rIFN 7 upregulated expression of both MHC class I and II molecules. To determine if this was a result of an increased level of specific mRNA, RNA was isolated from fresh macrophages, macrophages incubated for 24 h in RF10 alone, or macrophages incubated for 24 h in the presence of l0 U m l - 1 of rIFNT. The RNA was electrophoresed, blotted to a nylon membrane and hybridised consecutively with DNA probes specific for mRNA encoding MHC class I antigens, MHC class II antigens (DQA specific probe) and fltubulin. Results of a representative experiment (three donors) presented in Fig. 3 indicate that in both cases upregulation of surface expression was due to an increased level of specific mRNA. With regard to class II expression, results (Fig. 3A) show that in the absence of rIFN7 there was a dramatic decrease in the level of class II specific message after 24 h in culture. Incubation with rIFN7 resulted in a level of message, after 24 h, approximating the level observed in freshly isolated cells. In contrast, there was no obvious change in the level of class I specific message after 24 h in RF10 alone, however, there

84

A.D. NASH ET AL.

123

;

'q28S

A

418S

,~iiiiiiii~'f ~......

B

~

~i~

.~18S

Fig. 3. Northern blot analysis of the regulation of MHC class I and II expression by rlFNT. Ten micrograms of total RNA extracted from freshly isolated alveolar macrophages (lane 1 ), macrophages cultured for 24 h in RFI 0 (lane 2) or macrophages cultured for 24 h in RF10 supplemented with 10 U ml-1 of rlFN~, was hybridised consecutively with probes specific for MHC class II mRNA (A), MHC class I mRNA (B), and fl-tubulin mRNA (C). After washing, autoradiographic exposure was for 24 h. The position of the 28S and 18S ribosomal bands is indicated. was a significant increase in the level o f this message in the presence o f r l F N y (Fig. 3B).

Cytokine production by LPS stimulated alveolar macrophages Kinetics In the next series o f experiments we characterised the production of the cytokines IL-I a, IL-fl, and T N F a by resting and LPS stimulated alveolar macrophages of five sheep. At the molecular level, cross-species homology permitted us to use the comparable h u m a n c D N A probes to detect specific m R N A . With regard to biological activity only the production of IL-1 was readily characterised as ovine T N F a was not detected in cross-species bioassays (result not s h o w n ) . Macrophages were recovered from lung washings and cultured in R F 1 0 alone or in R F I 0 supplemented with 10/tg ml-1 o f LPS. Total R N A was extracted from these macrophages at various time intervals and also from freshly isolated cells. The R N A was electrophoresed, blotted to nylon m e m b r a n e s

OVINEALVEOLARMACROPHAGES

85

then hybridised with cDNA probes specific for human IL-1 t~, IL-1 fl, T N F a or control GAPDH and//-tubulin. None of the cytokine specific probes hybridised with RNA from freshly isolated macrophages (results not shown) or macrophages cultured in medium alone. In contrast, in results of a representative experiment shown in Fig. 4 transcripts encoding all three cytokines appeared at various time points following stimulation with LPS. The level of TNFtx specific mRNA peaked at 1 h after initiation of culture and declined thereafter with only a trace being detected at 24 h but none at 48 h (Fig. 4D). IL- 1~ specific mRNA was also detected after 1 h of stimulation with an in1 2345678910 A 428S 418S B 428S 418S C 1 23456 D ,428S ,~18S

E

Fig. 4. Northern blot analysis of induction of IL- I a, IL-I fl and TNFc~ specific mRNA by LPS. (A, B, and C) Ten micrograms of total RNA extracted from alveolar macrophages cultured for 30 rain (lanes 1 and 6), l h (lanes 2 and 7), 5 h (lanes 3 and 8), 24 h (lanes 4 and 9), and 48 h (lanes 5 and t0) in RF10 supplemented with 10 #g ml -t LPS (lanes 1-5) or RF10 alone (lanes 6-10) was hybridised consecutively with probes specific for IL- 1cz (A), IL- 1fl (B), and GAPDH (C). (D and E) Ten micrograms of total RNA extracted from alveolar macrophages cultured for I h (lanes 1 and 4), 5 h (lanes 2 and 5), and 24 h (lanes 3 and 6) in RFI0 supplemented with 10/~g ml- t of LPS (lanes 1-3) or RF10 alone (lanes 4-6) was hybridised consecutively with probes specific for TNF~x (D) and fl-tubulin (E). Autoradiographic exposure was for 24 h. The position of the 28S and 18S ribosomal bands is indicated.

86

A.D. NASH ET AL. JO'

A

llO.

4O

2O

O~

•

i

~

i

0

10

20

30

40

0 ~

w

;

: SO

|

i

0

10

20

30

40

SO

i

0

10

20

30

40

50

12000100008OOO

6OOO 4OOO

2OOO 0 T i m e (hr=)

Fig. 5. Time course of IL-1 secretion by LPS stimulated and unstimulated alveolar macrophages. Freshly isolated macrophages (A and B) or macrophages pre-cultured for 24 h (C) were cultured in RF10 (A) or RFI0 supplemented with I0 pg ml - t LPS (B and C). Culture SNF was sampled at 1 h, 5 h, 24 h and 48 h and assayed for IL-1 activity as described in Materials and Methods.

creased level observed after 5 h o f culture. No mRNA encoding this cytokine was detected at either 30 min stimulation or 24 or 48 h after stimulation (Fig. 4A). In contrast to IL-I ot message, mRNA encoding IL-1 fl was not detected

OVINEALVEOLARMACROPHAGES

A

87

30002600,

lSOO 1000

6OO

I

I

|

|

107 l0 s 105 104 10s 102 10 1

O.1

Kpglmt)

1234 B

C

Fig. 6. Dose response for induction of IL-I and TNFot by LPS. (A) Freshly isolated alveolar macrophages were cultured in RF10 supplemented with LPS. SNF was harvested at 24 h and assayed for IL-1. (B) Ten micrograms per track of total RNA extracted from alveolar macrophages stimulated for 1 h with 100 ng m l - ~ LPS (lane 1 ), 10 ng m l - t LPS (lane 2), 1 ng m l - i (lane 3 ) and 0.1 ng ml - ~ (lane 4) was hybridized consecutively with probes specific for T N F a (B) and fl-tubulin (C). Autoradiographic exposure was for 24 h.

until after 5 h of stimulation. A low level o f m R N A remained at 24 h but none was detected after 48 h stimulation (Fig. 4B). The kinetics of IL-1 secretion into the surrounding culture m e d i u m was determined using the N O B - 1 / C T L L assay. Macrophages were cultured in RF10 alone or in R F I 0 supplemented with I0 #g ml -~ LPS. SNF was sampled from these cultures after 1, 5, 24 and 48 h. Results show that in the absence of exogenous stimulation small amounts of IL- 1 were secreted into the SNF (Fig. 5A). No IL-1 was detected after 1 h culture, 5 U ml -~ was observed after 5 h and the level peaked at approximately 80 U m l - ~ after 24 h.

88

A.D. NASH ET AL.

To ensure that the secretion of IL-I in the absence of stimulation did not affect the determination of kinetics of induction following specific stimulation we studied secretion of IL-1 by freshly isolated macrophages as well as macrophages that had been pre-cultured for 24 h in RF10. Kinetics varied markedly for these two macrophage populations (Figs. 5B and 5C). In the presence of LPS the kinetics oflL-1 secretion by freshly isolated macrophages was similar to that observed for unstimulated macrophages, although the actual level of IL-I secreted was increased hy approximately 150-fold. A comparatively low level of IL-1 was detected after 5 h of culture (320 U ml -~ ) and this increased to approximately 10 000 U ml-~ after 24 h (Fig. 5B). In contrast, macrophages pre-cultured for 24 h secreted high levels of IL-1 within 5 h of addition of LPS and this level increased only slightly over the next 19 h. Dose response

A dose response curve for the secretion of IL-1 from macrophages of three donors following stimulation with LPS was determined using the NOB-1/ CTLL bioassay. Macrophages were cultured in RF 10 supplemented with 10fold dilutions of LPS from 10 mg ml-1 to 0.1 pg m1-1. SNF was sampled at 24 h and assayed for IL-1. Results of a representative experiment show that maximal IL- 1 secretion was induced with concentrations of LPS greater than 10 ng m l - ' (Fig. 6A ). At concentrations below this the level of IL- 1 secreted decreased until at 0.1 pg m l - ~ of LPS only background levels were detected. As this bioassay does not differentiate between IL-1 re and IL-1 fl a similar experiment was performed with RNA extracted from macrophages stimulated with different concentrations of LPS for 5 h. The RNA was hybridised with cDNA probes specific for IL-1 re and IL-1 ft. The level of detectable m R N A declined rapidly with concentrations of LPS less than 1 ng ml-~ for both probes, implying a similar dose response for both IL-1 re and IL-lfl (not shown). A similar Northern blot was used to determine the dose response for induction of TNFre by LPS. Results presented in Fig. 6B show that the level of detectable TNFre specific message declined over the dose range tested. DISCUSSION

Regulation of expression of MHC molecules plays a critical role in initiation and expression of the i m m u n e response, as T cell subpopulations generally recognise antigen either in the context of MHC class I or class II molecules (Moiler, 1988 ). For antigen presenting cells such as macrophages, class II expression together with the capacity to process antigen and release secondary accessory signals such as IL- 1 is an essential characteristic. IFN7 has been demonstrated to be a potent regulator of M H C antigen expression on h u m a n

OVINE ALVEOLAR MACROPHAGES

89

and murine macrophages (Kelley et al., 1984; Wong et al., 1984). The strict species specificity of IFN~, and lack of appropriate probes however has meant that study of this important regulatory mechanism in other species has been limited. Results presented in this report demonstrate that rbolFNT, which crossreacts with ovine cells, influences the level of MHC specific mRNA and, through this, the level of MHC antigen expressed on the ovine macrophage cell surface. The regulatory capacity of rlFN~, was well conserved when compared with human and murine models as indicated by similar kinetics and dose/response (Cao et al., 1989). Flow cytometric analysis indicated that greater than 10 U m l - ~ of rlFNy induced maximal expression of both class I and II molecules within 24 h and that this level persisted for at least a further 24 h. That this upregulation was caused by the addition of rlFN~, was verified by the blocking effect of rlFN7 specific mAbs. At the molecular level, the present results are consistent with observations made at the cell surface. Ten units per millilitre of rlFNy led to increased levels of both class I and class II specific mRNA within 24 h of stimulation. Studies in other species would suggest that this observed increase in specific mRNA was due to an increased rate of transcription rather than increased stability of existing message (Fertsch et al., 1984; Koerner et al., 1987). The absence of exogenous stimulation led to a rapid and substantial decline in the level of class II specific mRNA. For murine macrophages a rapid decline of surface Ia expression with time in culture has been reported (Belier and Unanue, 1981 ). In contrast to these studies we noted an obvious decline in the level of class II surface expression in only about 20% of lung macrophage populations tested (one of five donors). This may be due to a less rapid turnover of class II molecules on the ovine macrophage cell surface compared with the murine macrophage cell surface. In further studies not reported here we have examined the influence of rlFN7 on MHC expression on primary ovine fibroblasts. In contrast to results obtained with macrophages as target cells, incubation of fibroblasts with rlFN~, for 48 h led to a 10-100-fold increase in MHC class I expression but, at the cell surface at least, not induction of class II expression. This result, which was in agreement with previous observations (Wong et al., 1984) where it was noted that rlFN~, did not influence the level of class II antigen expressed on the surface of murine fibroblast lines, provides further evidence for the conservation of rlFNy bioactivity between the ovine model and murine and human models. Thus it is reasonable to assume that the spectrum of ovine cells on which MHC expression is regulated by IFN7 would include cells of hemopoietic, lymphoid, epithelial, and neuronal origin (Wong et al., 1984). In vitro exposure of human and murine macrophage populations to LPS has been demonstrated to induce production of IL- 1 and TNFot. This in vitro phenomenon is not without biological significance as in acute septicaemia

90

A.D. NASH ET AL.

LPS is the agent responsible for the increased serum levels of IL- 1 and T N F a that are associated with septic shock (Heremans et al., 1990). We examined production of IL-1 a, IL-1 fl, and T N F a by alveolar macrophages freshly isolated or cultured in the presence or absence of LPS. Using Northern blot analysis we failed to detect m R N A encoding these cytokines in total RNA from freshly isolated macrophages. Similarly, no specific m R N A was detected in total RNA from macrophages cultured in m e d i u m alone. This observation was in agreement with results obtained using various h u m a n and murine macrophage populations where, in the absence of exogenous pyrogen, little or no specific m R N A or secreted bioactive protein could be detected (March et al., 1985; Poole et al., 1989). Addition of LPS upregulated production of all three sheep cytokines. With regard to IL-1 this was reflected by increased levels of specific m R N A as well as increased extracellular bioactive protein. ILl a m R N A was induced more rapidly than IL-lfl mRNA, although IL-lfl m R N A persisted for an extended period. We have confirmed the dominance of IL- 1fl message with time after stimulation during cDNA cloning of ovine IL- 1a and IL- 1ft. In a library prepared from pooled RNA extracted from macrophages stimulated with LPS for 5 and 16 h the frequency of IL-1 fl clones was 20-fold greater than that of IL-1 a clones (or T N F a clones, results not shown). These kinetics of IL-1 m R N A induction were similar to those reported for human, murine and bovine macrophage populations (Maliszewski et al., 1988; Schindler et al., 1989 ). In these macrophage populations both ILl mRNAs peaked at 4-6 h and IL- 1fl message increased as a proportion with time after stimulation. Smith et al. ( 1989 ) noted that in h u m a n alveolar macrophages stimulated for 4 h the ratio of IL-lfl to I L - l a m R N A was 3.45:1. These studies did not record an earlier induction of IL-1 a message and there are reports that IL- 1fl protein is detected extracellularly many hours prior to IL-1 a. This however, was demonstrated to be the result of delayed processing/secretion of pro-IL-1 a rather than a difference in levels of transcription (Hazuda et al., 1988). Reports in h u m a n and murine models where kinetics of IL- 1 transcription have differed substantially from those reported here have used alternative inducers, such as PMA and IL-1 itself. These give rise to ILl m R N A species of greater stability (Fenton et al., 1988; Schindler et al., 1989) and this leads to the suggestion that there are at least two routes of induction of IL- 1 transcription. The kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages were as expected given the time of maximal levels of m R N A and the previously reported delay in processing and secretion of translated IL-1. In activated h u m a n monocytes IL- 1fl has an intracellular half life of 2.5 h while the delay in secretion of translated IL-1 a is up to 10 h (Hazuda et al., 1988 ). We detected low levels of IL-I in the SNF of macrophages cultured in medium alone. No IL-1 m R N A of either type was detected in total RNA extracted from these cells implying greater sensitivity of the bioassay. The ad-

OVINE ALVEOLAR MACROPHAGES

91

dition of LPS resulted in low levels of IL- 1 activity being detected at 5 h and dramatically increased levels at 24 h ( 10 000 U m l - 1). These kinetics were similar to those reported for other species (Hazuda et al., 1988; Beuscher et al., 1990). The kinetics of secretion of IL-1 were faster when macrophages were pre-incubated for 24 h prior to stimulation. As noted above the rate of secretion of bioactive IL- 1 is dependent on processing of precursor proteins as they pass through the cell membrane. The enzyme responsible for this processing is itself subject to induction by various stimuli (Beuscher et al., 1990) and it is possible that the kinetics we observed for pre-incubated macrophages were a result of upregulated processing. The dose response for induction of IL-1 by LPS was similar to previous reports (Poole et al., 1989; Schindler et al., 1989 ). Beuscher et al. (1990) noted that in elicited murine macrophages, concentrations of LPS less than 1/zg ml-~ induced secretion of only the unprocessed and biologically inactive form of IL- 1ft. They suggested that this was due to the failure of low LPS concentration to induce the appropriate processing enzymes. If applied to the data reported here this would suggest that although we have detected both IL-1 c~ and IL-1 fl mRNA induction at LPS concentrations well below 1/tg m l - ~, the bioactivity we detect in the SNF of these cells (Fig. 6 ) is due predominantly to IL-1 a activity. Kinetics for LPS induction of TNFot mRNA were more rapid than those of either IL- 1a or IL- 1fl although the dose response was similar. Maximum levels of TNFo~ mRNA were detected within 1 h of stimulation with the level declining thereafter and LPS concentrations as low as 10 ng m l - ~ were able to induce T N F a mRNA. These data were compatible with parameters of T N F a mRNA induction observed in macrophage populations of other species (Beutler et al., 1986; Kunkel et al., 1988; Schindler et al., 1989). Taffet et al. (1989) demonstrated that in a murine macrophage cell line maximum LPS induction of TNFol mRNA was at 1 h with significant levels remaining at 6 h. They also demonstrated that this increase was due to an increased rate of transcription rather than accumulation of message. The results presented in this study demonstrate that functional capacity of ovine alveolar macrophages, and thus probably other ovine macrophage populations, is subject to the same regulatory mechanisms demonstrated for macrophage populations in other species. Macrophage MHC antigen expression, critical for initiation of immune responses, was demonstrated to be regulated by IFNy and exposure of macrophages to LPS led to a rapid upregulation in the synthesis of the cytokines IL- I a, IL- 1fl and TNFo~. ACKNOWLEDGEMENTS

The authors gratefully acknowledge Immunex Research and Development Corporation, Genentech Inc, and Dr. P. Wood for providing cDNA probes, recombinant molecules, and monoclonal antibodies. We also acknowledge K.

92

A.D.NASHETAL.

S n i b s o n for p r e p a r a t i o n o f p h o t o g r a p h s . This s t u d y was s u p p o r t e d b y g r a n t U M 2 3 P f r o m the A u s t r a l i a n W o o l C o r p o r a t i o n .

REFERENCES Belier, D.I. and Unanue, E.R., 1981. Regulation of macrophage populations. II. Synthesis and expression of Ia antigens by peritoneal macrophages is a transient event. J. Immunol., 126: 263-269. Beuscher, H.U., Gunther, C. and Rollinghoff, M., 1990. IL-lfl is secreted by activated murine macrophages as biologically inactive precursor. J. Immunol., 144: 2179-2183. Beutler, B., Krochin, N., Milsark, I.W., Luedke, C. and Cerami, A., 1986. Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science, 232:977980. Cao, H., Wolf, R.G., Meltzer, M.S. and Crawford, R.M., 1989. Differential regulation of class II MHC determinants on macrophages by IFN-y and IL-4. J. Immunol., 143:3524-353 i. Chang, R.J. and Lee, S.H., 1986. Effects of interferon-y and tumor necrosis factor a on the expression of Ia antigen on a murine macrophage cell line. J. Immunol., 137: 2853-2856. Chomczynski, P. and Sacchi, N., 1987. Single step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162:156-159. Cleveland, D.W., Lopata, M.A., MacDonald, R.J., Cowan, N.J., Rutter, W.J. and Kirshner, M.W., 1980. Number and evolutionary conservation of a- and fl-tubulin and cytoplasmic fland y-actin genes using specific cloned cDNA probes. Cell, 20: 95-105. Dayer, J.M., Beutler, B. and Cerami, A., 1985. Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J. Exp. Med., 162: 2163. Ellis, J.A., Morrison, W.I., Goddeeris, B.M. and Emery, D.L., 1987. Bovine mononuclear phagocytes: identification by monoclonal antibodies and analysis of functional properties. Vet. Immunol. Immunopathol., 17:125-134. Fenton, M.J., Vermeulen, M.W., Clark, B.D., Webb, A.C. and Auron, P.E., 1988. Human proIL-lfl gene expression in monocytic cells is regulated by two distinct pathways. J. Immunol., 140: 2267-2273. Fertsch, D.D.R., Schoenberg, R.N., Germain, J.Y.L., Tou, L. and Vogel, S.N., 1984. Induction of macrophage Ia antigen expression by rIFN-y and down regulation by interferon-a/fl and dexamethasone are mediated by changes in steady state levels of Ia mRNA. J. Immunol., 139: 244-249. Gearing, A.J.H., Bird, C.R., Brostow, A., Poole, S. and Thorpe, R., 1987. A simple, sensitive bioassay for interleukin-1 which is unresponsive to 103 units/ml of interleukin-2. J. Immunol. Methods, 99:7-11. Harrison, L.C. and Campbell, I.L., 1988. Cytokines: An expanding network ofimmuno-inflammatory hormones. Mol. Endocrinol., 2:1151 - 1156. Hazuda, D.J., Lee, J.C. and Young, P.R., 1988. The kinetics of interleukin-1 secretion from activated monocytes. Differences between interleukin 1o~and interleukin 1ft. J. Biol. Chem., 263: 8473-8479. Heremans, H., Van Damme, J., Dillen, C., Dijkmans, R. and Billiau, A., 1990. Interferon-),, a mediator of lethal lipopolysaccharide induced Shwartzman-like shock reactions in mice. J. Exp. Med., 171: 1853-1870. Kampschmidt, R.F., 1984. The numerous postulated biological manifestations of interleukin1. J. Leukocyte Biol., 36: 341-355. Kelley, V.E., Fiers, W. and Strom, T.B., 1984. Cloned human interferon-y, but not interferon-fl

OVINE ALVEOLAR MACROPHAGES

93

or -a induces expression of HLA DR determinants by fetal monocytes and myeloid leukemic cell lines. J. Immunol., 132: 240-245. K o e r n e r , J.J., Hamilton, T.A. and Adam, D.O., 1987. Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA. J. Immunol., 139: 239-243. Koski, I.R., Poplack, D.G. and Blaese, R.B., 1976. In: B.R. Blood and J.R. David (Editors), In vitro Methods in Cell Mediated and Tumor Immunity. Academic Press, New York, p. 359. Kunkel, S.L., Spengler, M., May, M.A., Spengler, R., Larrick, J. and Renmick, D., 1988. Prostaglandin E2 regulates macrophage derived tumor necrosis factor gene expression. J. Biol. Chem., 253: 5380-5384. Mackay, C.R., Kimpton, W.G., Brandon, M.R. and Cahill, R.N.P., 1988. Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes. J. Exp. Med., 167: 1755-1766. Mackay, C.R., Maddox, J.F. and Brandon, M.R., 1986. Three distinct sub-populations of sheep T-lymphocytes. Eur. J. Immunol., 16: 19-25. Mackay, C.R., Maddox, J.F. and Brandon, M.R., 1987. A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset. Cell. Immunol., 110: 46-55. Maliszewski, C.R., Baker, P.E., Schoenborn, M.A., Davis, B.S., Cosman, D., Gillis, S. and Cerretti, D.P., 1988. Cloning, sequence, and expression of bovine interleukin-1 a and interleukin-lfl complementary DNAs. Mol. Immunol., 25: 429-437. March, C.J., Mosely, B., Larsen, A., Cerretti, D.P., Braedt, G., Price, V., Gillis, S., Henney, C.S., Kronhein, S.R., Grabstein, K., Conlon, P.J., Hopp, T.P. and Cosman, D., 1985. Cloning, sequence, and expression of two distinct human interleukin 1 complementary DNAs. Nature, 315: 641-647. Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J., Welgus, H.G. and Kuwano, M., 1982. Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors, and the production of tissue inhibitor or metalloproteinases and IL-6 in human microvascular endothelial cells. J. Immunol., 143:1619-1627. Meeusen, E., Barcham, G.J., Gorrell, M.D., Rickard, M.D. and Brandon, M.R., 1990. Cysticercosis: Cellular immune responses during primary and secondary infection. Parasite Immunol., 12: 403-418. Moller, G. (Editor), 1988. Antigen processing. Immunol. Rev., 106: 1-187. Oppenheim, J.J., Kovacs, E.J., Matsushima, K. and Durum, S.K., 1986. There is more than one interleukin 1. Immunol. Today, 7: 45-56. Piechaczyk, M., Blanchard, J.M., Many, L., Dani, C.H., Panabieres, F., Sabouty, S.E., Fort, P. and Jeanteur, P., 1984. Post transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression in rat tissues .Nucl. Acids Res., 12:6951-6963. Poole, S., Bristow, A.F., Selkirk, S. and Raffeny, B., 1989. Development and application of radioimmunoassays for interleukin- 1c~and interleukin- 1p. J. Immunol. Methods, 116: 259264. Puff, N.K., Gogolin-Ewens, K. and Brandon, M.R., 1987. Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct sub-populations of class II molecules. Vet. Immunol. Immunopathol., 15: 5986. Renz, H., Gong, J.H., Schmidt, G., Nain, M. and Gemsa, D., 1988. Release of tumor necrosis factor a from macrophages. J. Immunol., 141: 2388-2393. Sambrook, J., Fritsch, E.F. and Maniatis, T., (Editors), 1989. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Schindler, R., Gezzi, P. and Dinarello, C.A., 1989. IL-I induces IL-1. IV. IFN-y suppresses ILl but not lipopolysaccharide induced transcription of IL- 1. J. Immunol., 144:2216-2222.

94

A.D. NASH ET AL.

Scott, P.C., Choi, C.L. and Brandon, M.R., 1987. Genetic organisation of the ovine MHC class II region. Immunogenetics, 25:116-122. Smith, M.F., Kueppers, F.R., Young, P.R. and Lee, J.C., 1989. A rapid and quantitative method for the determination of interleukin-lce and interleukin-lfl mRNA expression in human monocytes and macrophages. J. Immunol. Methods, 118: 265-272. Stuart, R.M., Zlotnik, A. and Woodard, J.G., 1988. Induction of MHC class I and class I1 MHC antigen expression on bone marrow derived macrophages by IL-4 (B cell stimulatory factor 1 ). J. Immunol., 140: 1542-1547. Taffet, S.M., Singel, K.J., Overholtzer, J.F. and Shurtleff, S.A., 1989. Regulation of tumor necrosis factor expression in a macrophage like cell line by lipopolysaccharide and cyclic AMP. Cell. Immunol., 120: 291-300. Townsend, A. and Bodmer, H., 1989. Antigen recognition by class I restricted T-lymphocytes. Annu. Rev. Immunol., 7: 601-624. Weiss, E., Golden, L., Zakut, R., Mellor, A., Fahrner, K., Kvist, S. and Falvell, R.A., 1983. The DNA sequence of the H-2Kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibility antigens. EMBO. J., 2: 453-462. Wong, G.H.W., Clark-Lewis, I., Harris, A.W. and Sehrader, J.W., 1984. Effect of cloned interferon-? on expression of H-2 and Ia antigens on cell lines of hemopoietic, lymphoid, epithelial, fibroblastic and neuronal origin. Eur. J. Immunol., 14: 52-56. Wood, P.R., Rothwel, J.S., McWaters, P.D.G. and Jones, S.L., 1990. Production and characterization of monoclonal antibodies specific for bovine gamma-interferon. Vet. Immunol. Immunopathol., 25: 37-46.