Technical Advance - Europe PMC

American Journal of Pathologv, Vol. 147, No. 5, November 1995 Copynght C) Amencan Societyfor Investigative Pathology

Technical Advance Immunohistochemical Detection of Active Transforming Growth Factor-f3 in Situ Using Engineered Tissue

Mary Helen Barcellos-Hoff,* E. J. Ehrhart,t Manu Kalia,* Randolph Jirtle,t Kathleen Flanders,§ and Monica L.-S. Tsang" From the Life Sciences Division,* Lawrence Berkeley Laboratory, University of California, Berkeley, California; the Department of Radiological Health Sciences,t Colorado State University, Ft. Collins, Colorado; the Department of Radiation Oncology,t Duke University Medical Center, Durham, North Carolina; the National Cancer Institute,5 Laboratory of Chemoprevention, National Institutes of Health, Bethesda, Maryland; and R & D Systems, Inc.," Minneapolis, Minnesota

The biological activity of transforming growth

factor-fl) (TGF-f3) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-(3 could provide insight into TGF-f3 activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-f3 status is known. To provide in situ analysis of antibodies to differentiate between these functionalforms, we used xenografts of human tumor ceUs modified by transfection to overexpress latent TGF-j8 or constitutively active TGF-fk This comparison revealed that, whereas most antibodies did not differentiate between TGF-f3 activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-f8, LC(I-30) and CC(I-30) showed marked preferential immunoreactivity with active TGF-f8 versus latent TGF-f3 in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-f8 by CC(I-30) was lost and immunoreactivity was distinctly extracelu-

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lar, as previously reported for this antibody. Similar processing-dependent extracelular localization was found with a neutralizing antibody raised to recombinant TGF-fI Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-f8 only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-f in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-(3 depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with speciflcfunctinalforms of a protein (Amj Pathol 1995, 147:1228-1237)

Transforming growth factor-f (TGF-,B) is secreted as a latent complex (LTGF-,B) consisting of the TGF-,B homodimer noncovalently associated with a dimer of the processed amino-terminal pro-segment, which is called latency-associated peptide (LAP).1'2 Biological activity is controlled by activation, which occurs extracellularly when TGF-f3 is dissociated from LAP.3'- As a result, elevated expression of latent Supported by a NASA National Center for Research and Training in Radiation Health (MHBH and EJE) and the Office of Health and Environmental Research, Health Effects Research Division, of the U.S. Department of Energy contract DE-AC-03-76SF00098 (MHBH). Accepted for publication August 2, 1995. Address reprint requests to Dr. Mary Helen Barcellos-Hoff, Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Building 74, Rm. 157, 1 Cyclotron Road, Berkeley, CA 94720.

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complex may not have biological consequences, whereas increased activation, even without changes in synthesis, will profoundly affect physiological events.6 Activation has been postulated to occur in a wide variety of physiological and pathological situations in vivo.7 Most tissues express TGF-,B mRNA and protein,8 but biochemical extraction results in LTGF-,B activation,9 which has precluded biochemical analysis of endogenous levels of active TGF-4 in tissues. TGF-4 immunohistochemical localization poses several problems as a result. Antibodies have been widely used to localize proteins in tissues to better understand their role in complex physiological and pathological processes. However, interpretation of antibody staining is complicated as immunoreactivity may reflect either protein abundance or accessibility, both of which can be altered by tissue processing. Additional constraints exist if the antibody's ability to bind is determined by conformational characteristics of the target protein or is directed against a protein that has several functional forms. It is therefore important to test antibody specificity and define immunostaining conditions in a tissue in which the abundance of the target protein is known. When independent determination of protein access or distribution between functional forms is not technically feasible, these issues are not easy to resolve. Some TGF-, antibodies might be sensitive to the functional status of TGF-,B whereas others may be indifferent. TGF-4 antibody recognition may be impartial to activation if an antibody recognizes epitopes that are accessible in both protein forms. However, if the epitopes are masked, for example by association with proteins conferring latency, or if epitope recognition depends on protein conformation, which may change with activation, TGF-,B immunoreactivity might wholly depend on activation. As accurate immunolocalization also depends on target protein preservation after tissue fixation, concern about whether tissue processing preserves both LTGF-P and TGF-,B is a problem. The possibility that LTGF-f might be activated during tissue preparation has also been raised.10 The inability to independently ascertain the functional status of TGF-,B in situ has prevented analysis of these considerations. These technical limitations have precluded localizing sites of TGF-,3 activation and thus the events associated with or leading to biological activation of TGF-4 are not well understood. Pinpointing activation in situ may help resolve some questions surrounding the role of TGF-f3 in physiological and pathological processes.

Recent studies in our laboratory have suggested that ionizing radiation leads to TGF-f3 activation in vivo.11 12 Irradiated tissue exhibited rapid changes in TGF-13 immunoreactivity, which we suspected reflected activation-dependent antibody recognition. To confirm this interpretation, our strategy was to determine antibody specificity by using tissue specifically engineered to overexpress LTGF-,B and constitutively active TGF-j3.12 Directed mutation of two cysteines to serines in the LAP amino acid sequence results in secretion of predominantly active TGF-1.13 Sarcoma cells transfected with this construct or wildtype TGF-, produce tumors expressing active TGF-,3 and LTGF-,, respectively.14 We used this tissue to evaluate a panel of TGF-1 antibodies for their ability to discriminate TGF-,B from LTGF-f in cryosections or formalin-fixed, paraffin-embedded tumor sections. Although many antibodies did not differentiate between the active and latent forms of TGF-,B, several antibodies raised against synthetic peptides of the mature protein could differentiate, although this ability was dependent on tissue processing.

Materials and Methods Antibodies Purified polyclonal IgG fractions derived from rabbit antisera raised independently to synthetic TGF-/31 peptides corresponding to the amino-terminal 30 amino acids, LC(1-30) and CC(1-30),15 amino acids 78-109, D78-10916 and N78-1 09,17 and amino acids 50-75, N50-75,17 were examined. Also examined were pan-specific TGF-j1, -2, and -3 neutralizing rabbit antibody, RDnTGF-f3 (R & D Systems) and TGF-f3 neutralizing turkey antibodies, BD.654, BD.664, BD.414 (gift of Dr. F. Mannuzza, Becton Dickinson, Boston, MA). As immunoreactivity may be influenced by tissue composition, antigen abundance, and detection strategy, each antibody was individually titered on the tumor tissue for each fixation process used. Titration dilutions ranged from 1:10 to 1:200 (from 187 ,ug/ml to 10 ,ug/ml) and the concentration resulting in the most intense staining of tumor tissue was used as the working concentration (see Table 1).

Tumor Tissue Two clones of the human 293 renal sarcoma cell line transfected with TGF-,B expression plasmids were grown as xenografts.14 Site-directed mutagenesis of two cysteine codons to serine codons results in con-

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Barcellos-Hoff et al


stitutively active TGF-f3.2'13 Clone B9 produces LTGF-,B whereas clone C19, transfected with the mutated TGF-,B precursor, secretes predominantly active TGF-f3.14 Cells (2 x 106) were injected into each flank of adult female nude mice as described.12 Palpable tumors were obtained within 3 to 5 weeks, the mice were sacrificed by cervical dislocation in accordance with approved animal welfare guidelines, and the tumors removed for immunohistochemistry. The B9 and C19 tumors produce similar levels of total TGF-P as indicated by similar levels of LAP immunostaining.12

Immunohistochemistry Tumors were cut in half at the time of removal, and one-half was embedded in OCT compound (Baxter Scientific, McGaw Park, IL) and frozen in a dry ice/ ethanol bath. The other half was placed in 10% neutral-buffered formalin overnight, processed routinely with an automated processor, and embedded in paraffin. Bouin's fixation has been considered useful in TGF-f3 immunostaining,15 but preliminary data indicated that TGF-f3 immunoreactivity was severely compromised by this fixative in comparison with cryosections (data not shown). The frozen blocks were stored at -700C until sectioning; 4-,gm-thick sections were obtained by sectioning at -30 to -35°C. Two postfixations were compared for each antibody. Either the sections were immediately fixed onto gelatin-coated coverslips with -20°C methanol:acetone (1:1), air dried, and stored at -20oCll or unfixed,.frozen sections were thawed briefly, fixed for 20 minutes at room temperature with 2% paraformaldehyde in phosphate-buffered saline (Na2PO4, 0.9% NaCI) pH 7.4 (PBS), then rinsed three times with PBS containing 0.1 mol/L glycine. 12 Fixed cryosections were blocked for 60 minutes in supernatant from a solution of 0.5% casein in PBS (blocking buffer). Alternatively, formalin-fixed, paraffin-embedded sections were deparaffinized with xylene, rehydrated with graded alcohol, and washed in PBS before blocking. Pretreatment of formalin-fixed tissues with hyaluronidase (1 mg/ml testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) in 0.15 mol/L NaCI, pH 5.5 for 30 minutes) did not significantly alter immunoreactivity (data not shown). Antigen retrieval was used to process some formalinfixed paraffin sections before immunostaining. After deparaffinization and rehydration, the sections were placed in a vented plastic Coplan jar with 250 ml of antigen retrieval Citra solution (Biogenex, San Ramon, CA). The Coplan jar was set in a water bath

and microwaved for 5 minutes on high setting. The Citra solution level was readjusted and the sections were microwaved an additional 2 minutes. The slides were allowed to cool before resuming the immunostaining procedure. Sections were incubated with 25 ,al of the primary antibody diluted to working concentration in blocking buffer and incubated overnight at 40C. Antibody controls were incubated without primary antibody. Sections were washed and incubated with a 1:100 dilution of appropriate fluorescein isothiocyanate (FITC)-conjugated IgG secondary antibody for 1 hour at room temperature. Turkey antibodies were detected with biotin-conjugated secondary antibodies for subsequent avidin-FITC detection. Immunofluorescent sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Some paraffin sections were stained with avidin-biotin complexhorseradish peroxidase (Vector) and nickel-enhanced diaminobenzidine. Sections were then counterstained with Mayer's hematoxylin, dehydrated, and mounted with Permount (Fischer Scientific, Pittsburgh, PA). Sections were viewed with an Olympus microscope equipped with epifluorescence and photographed with Kodak Tri-X 400 color slide film. Each antibody was stained in duplicate in two independent experiments. To facilitate comparisons of a given antibody, batch-stained sections were photographed with identical exposures and the resulting transparency was scanned into digital format and printed with identical parameters.

Results Human 293 renal sarcoma cells transfected with mutant and wild-type constructs of TGF-,f were used to produced tumor xenografts.14 B9 designates a tumor cell clone stably transfected with the wild-type construct, which produces LTGF-1, and C19 is transfected with a mutated construct that produces constitutively active TGF-1. TGF-,3 immunoreactivity was evaluated in situ by comparing these tumors. All antibodies were initially tested by immunofluorescence in tumor cryosections after postfixation with either methanol:acetone or paraformaldehyde

(Table 1). A clear preference for active TGF-I3 was observed with the peptide antibody CC(1-30) with methanol/ acetone-fixed cryosections (Figure 1). We previously reported that LC(1-30), which was generated against the same peptide sequence, is specifically reactive with active TGF-13.12 As both antibodies recognize epitopes of active TGF-f3 but do not react with

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Table 1. Immunoreactivity of LTGF-13- and TGF-f3-Producing Tumors

Antibody

LC(1-30) CC(1-30) RDnRb N(78-109) D(78-109) N(50-75)

BD.654 BD.664 BD.414

Paraffin-embedded

Frozen

Concentration

LTGF-,B

TGF-f3

+ ++*

++ ++*

++ ++

++

++

+

+ ++ +

+ ++ +

+/-

++ ++

++ ++

++ ++

++ + ++ + ++ ++

(ag/ml)

LTGF-,B

TGF-f

125 82 50 187 72 100 84 107 83

+-

++

++ ++

++

++ +

Immunofluorescence intensity was graded on a 3+ scale: +, mild; ++, moderate; +++, marked. *Specific extracellular staining distribution.

LTGF-f3 in B9 tumors, these data suggest that their epitopes located in the first 30 amino acids of mature TGF-,B, or some portion thereof, are masked in the latent complex in situ. Previous characterization by Western blots and enzyme-linked immunosorbent assay demonstrated that both antibodies react with the mature/active 24-kd TGF-P.15 LC(1-30) immunoreactivity has been reported to be cell associated in Bouin's-fixed tissues, 15'1-20 formalin-fixed tissues,21'22 and frozen,

postfixed tissue.11"12 CC(1-30) is reported to show distinct extracellular distribution in paraffin embedded tissues, 15,18-21,23 although intracellular staining has been noted in human suprabasal keratinocytes.19 CC(1-30) preferential immunostaining of C19 tumor was predominantly cell associated in cryosections but was somewhat more intense in areas of extracellular matrix accumulation (Figure 1). In contrast, six other antibodies at dilutions ranging from 1:10 to 1:200 did not show preferential

Figure 1. Comparison of CC 1-30), RDnTGF-(3 immunoreactivity in cryostat sections. Tumor xenografts expressing LTGF-13 (A and C) and TGF-,3 (B and D) were stained with CC( 1-30) (A and B), and RDnTGF-,f (C and D). CC( 1-30) showed preferential immunoreactivity uwith TGF-,B whereas RDniTGF- / was equally reactive with LTGF-f3 and TGF-,B. The majority of the staining observed was cell associated, but CC(1-30) and RDnTGF-13 showed mildly enhanced staining of tissue septa (arrows). Controls had minimalfluorescence. Bar, 10 ,um.

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AJP November 1995, Vol. 147, No. 5

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Figure 2. Comparison of LC(1-30) immunostaining in formalin-fixed, paraffin-embedded sections. LTGF-4-producing tumors (B and E) and TGF-j3-producing tumors (C and F) were stained with L?1-30) by immunofluorescence (B and C) or peroxidase (E and F). Both detection techniques show distinct intracellular staining of tumor cells with preferential stainintg of the C19 tumor that producces TGF-3. Sonice extracelluclar staining is observed in the tumor capsule. Control sections (A and D) showed minimal staining. m, muscle; c, tumor capsule. Bar, 10 jim.

reactivity in cryosections with either methanol/acetone or paraformaldehyde postfixation. This pattern is demonstrated in Figure 1, C and D, with a TGF-,B neutralizing antibody, RDnTGF-p, which

was

raised

to recombinant 24-kd protein. RDnTGF-k exhibited moderately intense, cell-associated immunoreactivity in both B9 and C19 tissue sections. This cellassociated pattern is similar to RDnTGF-j3 localization reported with ethanol fixation.24 Like CC(1-30), RDnTGF-/ also showed mild increased immunoreactivity associated with the extracellular matrix of septae in cryostat sections.

Antibody immunoreactivity in cryosections was then compared with formalin-fixed, paraffin-embedded tumor sections. As seen with cryosections, LC(1-30) demonstrated preferential reactivity with the active TGF-f produced by the C19 tumor. Localization of this antibody in formalin-fixed sections was similar to that found in cryosections, although somewhat reduced in intensity. Distinct cellular reactivity was observed in tumor cells with either indirect FITC detection or biotin-avidin-amplified peroxidase detection and was also noted in the host cells of the fibrous capsule (Figure 2).

Immunodetection of Active TGF-,B in Situ 1233 AJP November 1995, Vol. 14 7, No. 5

Figure 3. Comparison of CC( 1-30) and RDnTGF-( antibodies in formalin-fixed, paraffin-embedded sections. LTGF-,B-producing tumors (A and D) and TGF-,(-producing tumors (B, C, E, and F) were stained witb C( 1-30) (A-C) and RDnTGF-P (D-F) by immunofluorescence with (C and F) and without (A, B, D, and E). antigen retrieval. No differential staining of TGF-(3 or LTGF-( was noted with either antibody, and both antibodies sbowed distinct extraceltular localization to tissue septae in formlin-fixed, paraffin-embedded sections, which contrasts with the intracellular immunoreactivity found in cryosections (Figure 1). Cell-associated immunoreactivity was recovered when sections were pretreated for antigen retrieval. Control sections bad minimalflutorescence (not shown). Bar, 10 /tm.

In contrast, CC(1-30) preferential irnmunoreactivity was lost in formalin-fixed, paraffin-embedded tumors, and cellular immunoreactivity was greatly reduced (Fi.gure 3). Extracellular immunoreactivity found in association wi'th the extracellular matrix and tissue septa was. strongly increased in paraffin sections. This immunostaining pattern is similar to previous reports noting extracellular localization by 0C(1-301) with formalin-fixed, paraffin-embedded tissue with'815 or without21 22 Bouin's fixative. In addition, iirmunostaining with the RDnTGF-,6 antibody also showed loss of cellular immunostaining and

distinct extracellular distribution. In both cases, the extracellular immunoreactivity was emphasized by the apparent loss of cellular staining found in cryosections. Remarkably, cell-associated RDnTGF-,B or CC(1-30) immunostaining was recovered by pretreatment of paraffin sections with antigen retrieval (Figure 3, C and F). However, after antigen retrieval, C0(1-30) failed to demonstrate preferential staining of active TGF-3-producing tumor sections. Two antibodies raised to synthetic peptide sequences 78-109 of the amino terminal of mature TGF-B, N(78-109) and D(78-109), showed en-

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Figure 4. Comparison of A( 78-109) immunoreactivity in cryosections and formalin-fixed, paraffin-embedded sections. LTGF-(3-producing tumors (A and C) and TGF-f3-producing tumors (B and D) were stained with A( 78-109) by immunofluorescence. In cryosections (A and B), A( 78-109) immunoreactivity was independent of TGF-f3 activation status, but informalin-fixed sections (C and D), moderate preference for active TGF-/3 was observed. In tumor parenchyma, a cell-associated staining pattern was predominant. n, nerve; v, autofluorescent vessel. Bar, 10 ,nm.

hanced differential staining of the tumor sections after formalin fixation (Figure 4). N(78-109) exhibited moderate preference for active TGF-p compared with latent TGF-,B, whereas D(78-109) exhibited a mild preference. As these antibodies did not exhibit preferential immunoreactivity in B9 and C19 cryosections, this processing-induced differential suggests that epitopes were exposed in the C19 tumor, or masked in the B9 tumor, by either the fixative or paraffin embedding. The other antibodies tested did not discriminate between active TGF-, and LTGF-f produced in the tumor xenografts under either tissue processing protocol. (Table 1).

Discussion The interpretation of immunoreactivity in tissue is confounded by the relative, and often unknown, contributions of protein abundance, access, and conformation, as well as antibody affinity. We propose that cell lines transfected to secrete specific forms of a particular protein that are grown as tumors offer a unique situation to characterize antigen recognition in situ. By using tumors engineered to express constitutively active TGF-P or

LTGF-4, we compared the immunoreactivity of various TGF-4 antibodies in either unfixed tumor cryosections that were postfixed with two different fixatives or sections of formalin-fixed tissue embedded in paraffin. As TGF-,B biological activity is controlled by extracellular activation, the ability to assign an antibody's relative preference for LTGF-,B or active TGF-,B may reveal new features of TGF-, localization, such as sites of activation. Two peptide antibodies to the TGF-,B amino terminus, LC(1-30) and CC(1-30), preferentially recognize active TGF-3 in cryosections. As TGF-f3 is part of the latent complex, it may be that these antibodies recognize epitopes that are masked by the association of TGF-3 with LAP or that they recognize a three-dimensional conformation conferred by activation. This interpretation is supported by the observation that both antibodies also exhibit increased immunoreactivity in cryosections of mouse mammary gland after ionizing radiation-induced activation.1112 Synthetic peptide antibodies raised to other regions of the mature TGF-,B that we screened did not reveal preferential immunoreactivity with active TGF-f in cryosections, which suggests that these epitopes are not affected by association with LAP.

Immunodetection of Active TGF-f in Situ

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We also compared cryosections with formalinfixed, paraffin-embedded tissue sections. Frozen, hydrated sections should preserve an antigen in a state close to its physiological status. Formalin preserves tissue morphology by creating protein crosslinks, which can also affect protein conformation and accessibility or availability for antibody binding. Protein associations, such as latency conferred by the association of TGF-,B with LAP, can also be altered by the fixative, solvents, or heat used during paraffin processing. Although most antibodies exhibited some degree of decreased immunoreactivity in formalin-fixed sections, N(78-109) and D(78-109) antibodies differentiated the active form of TGF-,B only after formalin fixation, perhaps by virtue of a conformational change in LTGF-,B that reduces access to this peptide region. This study sheds some light on the conundrum of why two antibodies, LC(1-30) and CC(1-30), raised against the same peptide sequence, produce such different immunolocalization. It has been suggested that cell-associated LC(1-30) immunoreactivity indicates the site of synthesis and that extracellular CC(1-30) localization is caused by conformational changes upon secretion.15 In this study, LC(1-30) immunoreactivity in formalin-fixed sections was cell associated whereas CC(1-30) exhibited specific extracellular localization as previously reported."15,18,23 But the preferential immunoreactivity that CC(1-30) demonstrates in tumor cyrosections is predominantly cell-associated, albeit with mild preference for extracellular matrix. Furthermore, CC(1-30) differential staining of active TGF-f3 and LTGF-, was lost in formalin-fixed sections. Likewise, RDnTGF-,B, although unable to discriminate between active and latent TGF-,B, also showed a pattern of cellular immunoreactivity in cryosections and extracellular staining in formalin-fixed sections. TGF-,B was not lost during formalin fixation or paraffin embedding since cellular immunoreactivity was detected with LC(1-30) and other antibodies. This was confirmed by antigen retrieval, which restored both CC(1-30) and RDnTGF-3 cell-associated immunoreactivity. Thus, either formalin or paraffin processing appears to mask distinct cell-associated CC(1-30) epitopes that are recovered with additional processing by antigen retrieval, but such processing also eliminates the ability of CC(1-30) to discriminate between active and latent TGF-,B. The intracellular versus extracellular localization of LC(1-30) versus CC(1-30) in formalin-fixed, paraffin-embedded tissue appears to be an artifact of tissue processing. We have recently demonstrated that ionizing radiation induces a rapid increase in LC(1-30) immuno-

reactivity concomitant with loss of LAP immunoreactivity in murine mammary gland that we propose is evidence of LTGF-,B activation in situ. 12 The ability to localize active TGF-,B provides a means for temporal evaluation of LTGF-, activation in physiological and pathological processes. Our present studies indicate that LC(1-30) is specifically immunoreactive with active TGF-f in both cryosections or formalinfixed, paraffin-embedded tissue. Thus LC(1-30) immunoreactivity may represent sites of LTGF-f3 activation in macrophages in atherosclerotic lesions,25 fibroblasts adjacent to hyperplastic epithelium in pulmonary fibrosis,22 suprabasilar cells at wound mar-

gins,'o papilloma epithelium,21 proliferative chondrocytes of ossifying bone,15 and regressing mammary tumor.26 The immunoreactivity of other extracellularly modified proteins might be evaluated with genetically engineered tissue. Growth factors, such as interleukin-1 and basic fibroblast growth factor, require extracellular modifications to evoke a tissue response.627 The delicate balance between tissue proteases, their inhibitors, and sites of activation cannot as yet be identified in situ with immunohistochemistry because many proteases, as well as their inhibitors and activators, are proenzymes. Determinants of immunoreactivity can also be influenced by interactions between proteins in the extracellular milieu. Although tumors are easily produced from engineered cell lines, they may not represent a completely normal environment. This problem could be rectified by using transgenic tissues that express the modified protein in a physiological environment. In conclusion, interpretation of immunostaining must be tempered until the antibody's in situ immunoreactivity has been thoroughly characterized. This paper introduces a technique to characterize an antibody's immunoreactivity with specific protein forms in situ. Our application of this strategy to TGF-f3 immunoreactivity underscores significant variation in detection depending on the specific antibody, tissue preparation, and tissue fixation. However, such evaluation has enabled us to determine that certain antibody preparations can specifically discriminate between active and latent forms of TGF-f. These antibodies will provide powerful tools for deciphering the role of TGF-j3 in tissue processes.

Acknowledgments The authors thank Dr B. Powers for reading the manuscript, Dr. F. J. Mannuzza for providing antibodies, and S. Ravani for technical assistance.

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Financial Disclosure Monica L.-S. Tsang is an employee of R & D Systems which has a financial interest in the RDnTGF-,B antibody used in this study.

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12. Barcellos-Hoff MH, Derynck R, Tsang ML-S, Weatherbee JA: Transforming growth factor-,8 activation in irradiated murine mammary gland. J Clin Invest 1994, 93:892-899 13. Brunner AM, Marquardt H, Malacko AR, Lioubin MN, Purchio AF: Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor ,13 precursor. J Biol Chem 1989, 264:13660-13664 14. Arrick BA, Lopez AR, Elfman F, Ebner R, Damsky CH, Derynck R: Altered metabolic and adhesive properties and increased tumorigenesis assoicated with increased expression of transforming growth factor ,B1. J

Cell Biol 1992, 118:715-726 15. Flanders KC, Tompson NL, Cissel DS, Van Obberghen-Schilling E, Baker CC, Kass ME, Ellingsworth LR, Roberts AB, Sporn MB: Transforming growth factor-,B1: histochemical localization with antibodies to different epitopes. J Cell Biol 1989, 108:653-660 16. Jirtle RL, Carr BI, Scott CD: Modulation of insulin-like growth factor-1l/mannose 6-phosphate receptors and transforming growth factor-1l during liver regeneration. J Biol Chem 1991, 266:22444-22450 17. Flanders KC, Roberts AB, Ling N, Fleurdelys BE, Sporn MB: Antibodies to peptide determinants in transforming growth factor 13 and their applications. Biochemistry 1988, 27:739-746 18. Sellheyer K, Bickenbach LAR Jr, Bundman D, Longley MA, Krieg T, Roche NS, Roberts AB, Roop RR: Inhibition of skin development by overexpression of transforming growth factor ,13 in the epidermis of transgenic mice. Proc NatI Acad Sci USA 1993, 90:5237-5241 19. Kane CJM, Knapp AM, Mansbridge JN, Hanawalt PC: Transforming growth factor-,1l localization in normal and psoriatic epidermal keratinocytes in situ. J Cell Physiol 1990, 144:144-150 20. Kane CJM, Hebda PA, Mansbridge JN, Hanawalt PC: Direct evidence for spatial and temporal regulation of transforming growth factor 13 expression during cutaneous wound healing. J Cell Physiol 1991, 148:157-173 21. Fowlis DJ, Flanders KC, Duffie E, Balmain A, Akhurst J: Discordant transforming growth factor 131 RNA and protein localization during chemical carcinogenesis of the skin. Cell Growth Differ 1992, 3:81-91 22. Williams AO, Flanders KC, Saffiotti U: Immunohistochemical localization of transforming growth factor-,1l in rats with experimental silicosis, alveolar type 11 hyperplasia, and lung cancer. Am J Pathol 1993, 142: 1831-1840 23. Broekelmann TJ, Limper AH, Colby TV, McDonald JA: Transforming growth factor 13 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991, 88:6642-6646 24. Pierce GJ, Mustoe TA, Lingelbach J, Masakowski VR, Griffin GL, Senior RM, Deuel TF: Platelet-derived growth factor and transforming growth factor-13 enhance tissue repair activities by unique mechanisms. J Cell Biol 1989, 109:429-440 25. Bahadori L, Milder J, Gold L, Botney M: Active mac-

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