By
From the * Dermatology Branch and the Laboratory of Chemoprevention, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892-1908; and the § Department of Biological
Structure, University of Washington, Seattle, Washington 98195
Transforming growth factor 1 (TGF-
1) regulates leukocytes and epithelial cells. To determine whether the pleiotropic effects of TGF-
1, a cytokine that is produced by both keratinocytes and Langerhans cells (LC), extend to epidermal leukocytes, we characterized LC (the
epidermal contingent of the dendritic cell [DC] lineage) and dendritic epidermal T cells
(DETC) in TGF-
1 null (TGF-
1
/
) mice. I-A+ LC were not detected in epidermal cell
suspensions or epidermal sheets prepared from TGF-
1
/
mice, and epidermal cell suspensions were devoid of allostimulatory activity. In contrast, TCR-
+ DETC were normal in
number and appearance in TGF-
1
/
mice and, importantly, DETC represented the only
leukocytes in the epidermis. Immunolocalization studies revealed CD11c+ DC in lymph nodes
from TGF-
1
/
mice, although gp40+ DC were absent. Treatment of TGF-
1
/
mice
with rapamycin abrogated the characteristic inflammatory wasting syndrome and prolonged survival indefinitely, but did not result in population of the epidermis with LC. Thus, the LC
abnormality in TGF-
1
/
mice is not a consequence of inflammation in skin or other organs, and LC development is not simply delayed in these animals. We conclude that endogenous TGF-
1 is essential for normal murine LC development or epidermal localization.
Transforming growth factor- The epidermal microenvironment is TGF- Mice.
TGF- Preparation of Epidermal Cell Suspensions and Epidermal Sheets.
Epidermal cell suspensions were prepared from trunk skin by limited trypsinization (0.25% trypsin [USB, Cleveland, OH] in HBSS without calcium or magnesium at 4°C for 18 h) and trituration in HBSS, 0.05% DNase, 30% FCS. Epidermal sheets were
prepared from ear skin by incubation in 0.5 M ammonium thiocyanate (37°C for 20 min), fixed in acetone ( Flow Cytometry and Immunofluorescence Microscopy.
Hybridomas
secreting mAb Y3-P (anti-I-Ab), MK-D6 (anti-I-Ad), and N418
(anti-CD11c) were obtained from American Type Culture Collection (Rockville, MD). G8.8 (anti-gp40) was produced and characterized as described (15). mAbs Y3-P, MK-D6, and N418 were purified from hybridoma supernatants by protein A affinity chromatography (Pierce Chemical Co., Rockford, IL); G8.8 was
purified using protein G (Pierce Chemical). These mAb were modified with FITC (Sigma) or NHS-LC-biotin (Bio; Pierce Chemical) as described (16). The following mAbs were purchased from
PharMingen (San Diego, CA): FITC-anti-TCR- (TGF-
) is comprised of
three isoforms (
1,
2, and
3) that are frequently coexpressed, that bind to the same receptor complex, and that
initiate similar biochemical changes in target cells in vitro
(1, 2). Thus, it is surprising that the phenotype of the homozygous null mutant of the murine gene encoding one of
these proteins, TGF-
1, is catastrophic. Although 50% of
TGF-
1
/
and 25% of TGF-
1+/
embryos experience prenatal demise, at birth TGF-
1
/
mice are normally formed and indistinguishable from +/+ and +/
littermates until postnatal d 10 (3). Thereafter, TGF-
1
/
mice exhibit a progressive wasting syndrome that results
from inflammation involving several organs that invariably
eventuates in death by postnatal d 30 (4, 5). The inflammation in TGF-
1
/
mice is lymphocyte and macrophage predominant, involves lungs, heart, liver, and other organs
(4, 5), and is associated with production of various autoantibodies (6, 7). Abnormalities in cytokine production (3),
MHC antigen expression (8), and nitric oxide metabolism
(9) have also been identified in TGF-
1
/
mice, but
the relationship of these findings to the genesis of the phenotype is uncertain.
1 rich; Langerhans cells (LC) as well as keratinocytes produce this pleiotropic cytokine (10). Previous studies of TGF-
1
/
mice demonstrated that TGF-
1 plays an important role in
epidermal homeostasis. Keratinocytes of TGF
1
/
mice
are hyperproliferative in vivo as well as in vitro, and are
predisposed to undergo malignant transformation (10, 13). To
determine whether abnormalities in epidermal homeostasis
extend to resident leukocytes in skin, we characterized epidermal LC and dendritic epidermal T cells (DETC) in
TGF-
1
/
mice. Our results implicate TGF-
1 as a
critical endogenous regulator of murine epidermal LC.
1
/
and littermate control (+/+) mice
were derived from matings of mice that were heterozygous for a
null mutation of the TGF-
1 gene (generated [4] and supplied by
A. Kulkarni and S. Karlsson [National Institute of Neurological
Disorders and Stroke, Bethesda, MD]). Since their derivation,
heterozygous males have been backcrossed to C57BL/6 females
four times and the colony has been maintained by interbreeding.
Thus, although mice in this colony express only H-2b MHC antigens, each animal has a mixture of 129Sv and C57BL/6 background genes and is unique. TGF-
1
/
and littermate control mice used in these studies were reared in a pathogen-free facility and were used at 8-20 d of age. All animals were housed and used in experiments in accordance with institutional guidelines.
20°C for 30 min),
and rehydrated in PBS (14).
(H57-597),
FITC-anti-TCR-
(GL3), FITC-Thy-1.2 (30-H12), Bio-anti-CD45 (30F11.1), and relevant isotype controls. Phycoerythrin-
streptavidin (PE-SA) was purchased from TAGO, Inc. (Burlingame, CA).
II [17]) followed by FITC-mAb,
Bio-mAb, and PE-SA. Surface antigen expression was analyzed
using a FACScan® flow cytometer equipped with Research Software (Becton Dickinson, Mountain View, CA). Propidium iodide permeable cells were excluded by live gating. Epidermal
sheets were stained for LC and DETC with FITC-anti-I-A or
FITC-anti-TCR mAb diluted in PBS/FCS/NaN3, washed, and
analyzed by epifluorescence microscopy.
Primary Allogeneic Reactions.
Epidermal cells derived from trunk
skin of TGF-1
/
and control mice were cocultured in flatbottomed 96-well plates with 2 × 105 accessory cell-depleted T cells
prepared from the skin-associated lymph nodes of female BALB/c
mice (18) for 120 h at 37°C. [3H]TdR (1 µCi/well) was added
for the final 12 h of the culture period. Cell-associated radioactivity was determined by direct
counting.
Immunohistochemistry.
Cells reactive with various lineageselective mAbs were localized in lymph nodes using a three-step
immunohistochemical procedure (19). In brief, acetone-fixed
frozen sections of tissues were washed with PBS and incubated
with hybridoma supernatants. After washing, mAbs were detected using digoxigenin-modified anti-rat (or anti-hamster) IgG
Ab (Pierce Chemical), peroxidase-conjugated sheep anti-digoxigenin Fab, and 3,3-diaminobenzidine. Digoxigenin-3-O-methylcarbonyl-
-aminocaproic acid, N-hydroxy-succinimidyl ester,
and anti-digoxigenin Ab were purchased from Boehringer Mannheim (Indianapolis, IN) and used as suggested by the manufacturer.
Rapamycin Treatment.
Rapamycin (Wyeth-Ayerst; Princeton,
NJ) was dissolved in 0.2% high viscosity carboxymethyl cellulose
(Sigma)/0.25% polysorbate 80 in dH20 and administered to all
progeny resulting from the mating of several breeding pairs of
TGF-1+/
mice by i.p. injection (4 mg/kg) on postnatal day
10 and 3×/wk thereafter.
To begin
to characterize epidermal leukocytes in TGF-1
/
mice,
cells were prepared from the trunk skin of
/
and +/+
littermates and examined for the simultaneous expression of
CD45 and I-A antigens, and CD45 and TCR-
(or Thy1.2) via multicolor flow cytometry. Although TGF-
1 +/+
mice contained a normal contingent of LC (CD45+ I-Ab+
cells), LC could not be identified in TGF-
1
/
epidermis
(see Fig. 1 for results with 17-d-old mice). However, the
frequency of DETC (CD45+ TCR-
+ cells) among
TGF-
1
/
epidermal cells was normal. In addition, essentially all of the leukocytes (CD45+ cells) present in
TGF-
1
/
epidermis were DETC. This latter result
excludes the existence of a significant population of immature LC (CD45+ I-Ab
TCR-
cells) in TGF-
1
/
epidermis. The CD45
I-A+ cells detected in TGF-
1
/
epidermis (see Fig. 1 a) represent keratinocytes that inappropriately express class II MHC antigens; this finding is
consistent with the previous report of disordered MHC Ag
expression in TGF-
1
/
mice (8). The LC deficiency in TGF-
1
/
mice was confirmed in six additional experiments with mice ranging in age from 7-18 d (Table 1).
Younger animals were not studied because normal numbers of epidermal LC would not be expected before postnatal day 7. Older animals were not studied because the
health status of TGF-
1
/
mice deteriorates rapidly after 18 d.
LC were also quantitated in TGF-1
/
and wildtype epidermis in situ. Immunofluorescence microscopy of
epidermal sheets prepared from the ear skin of TGF-
1
/
mice confirmed that LC were absent; DETC were normal
in number and morphology (data not shown). In addition,
epidermal cell suspensions from TGF-
1
/
mice failed to initiate a proliferative response in naive BALB/c T cells
(Fig. 2). Thus, LC (or LC precursors) could not be detected in the epidermis of TGF-
1
/
mice, or in epidermal cell suspensions prepared from these animals, using
assays of LC surface phenotype or function.
Lymphoid Dendritic Cells in TGF-
LC represent the epidermal contingent of the dendritic cell (DC)
lineage (20). To determine whether lymphoid DC were
present in TGF-1
/
mice, we examined skin-draining
lymph nodes for several leukocyte subpopulations. Axillary
lymph nodes were sectioned and stained with several lineage-selective mAbs (see Fig. 3). CD11c+ (N418-reactive)
DC were readily identified in T cell-dependent regions of
lymph nodes from TGF-
1
/
as well as control mice. In contrast, cells reactive with anti-gp40 (G8.8), another
DC-selective mAb (21), were absent from TGF-
1-deficient lymph nodes. F4/80+ macrophages were similarly
represented and distributed in both TGF-
1
/
and
TGF-
1 +/+ tissue. These results suggest that although lymphoid DC are present in TGF-
1 null mice, all subpopulations may not be normally represented. Alternatively, TGF-
1 may be required for expression of certain
lymphoid DC differentiation antigens.
Dissociation of the Langerhans Cell Deficiency from Other Aspects of the TGF-
The TGF-1
/
phenotype is characterized by marked inflammation of several
major organs, wasting, and death at an early age (3, 4). The
skin is not prominently involved in this inflammatory syndrome. Histopathologic examination of 17 d TGF-
1
/
skin revealed only a sparse interstitial inflammatory infiltrate that was difficult to distinguish from the minimal infiltrate present in controls (data not shown). Nonetheless, to
exclude the possibility that aberrant cytokine secretion associated with inflammation (in skin or elsewhere) influenced the ability of LC (or LC precursors) to develop or
localize in TGF-
1
/
epidermis, we assessed the LC
content of the skin of TGF-
1
/
mice that were
treated with the immunosuppressive rapamycin (22). Rapamycin (4 mg/kg) was administered i.p. to littermates
born to TGF-
1 heterozygotes beginning at 10 d of age
and 3×/wk thereafter. This regimen obviated the wasting
that is invariably present in untreated TGF-
1
/
mice
older than 14 d (Fig. 4), and almost completely inhibited the inflammation in lungs, heart, and liver that is characteristic of TGF-
1
/
mice (data not shown).
Rapamycin-treated TGF-1
/
mice not only survived indefinitely, but gained weight for the duration of
the experiments (39 and 42 d). Although the most dramatic
components of the TGF-
1
/
phenotype were eliminated by rapamycin treatment, epidermal sheets from ears
of rapamycin-treated TGF-
1
/
mice were devoid of
LC (data not shown), as were epidermal cell suspensions
prepared from trunk skin of these mice (Table 2). Rapamycin treatment had no effect on LC in TGF-
1 +/+ or +/
mice and, as in the case of untreated mice, DETC were the
only leukocytes present in appreciable numbers in TGF-
1
/
epidermis. Thus, the LC deficiency in TGF-
1
/
mice does not result from the inflammatory syndrome that
causes the demise of these animals, and cannot be attributed
simply to delayed development.
|
TGF-1
/
mice represent the first animal model in
which an absolute deficiency of LC (or other nonlymphoid
DC) has been identified. Decreased numbers of LC were
previously documented in the op/op mouse (23), a null
mutant of the M-CSF (CSF-1) gene, but the LC deficiency
in the TGF-
1
/
mouse is much more profound. Although the LC deficiency is the most obvious alteration in
the DC lineage in TGF-
1
/
mice, lymphoid DC may
also be abnormal. DC expressing gp40 (15, 21) were absent
from skin-draining lymph nodes of TGF-
1
/
mice,
whereas CD11c+ DC were readily detected. Perhaps gp40-bearing DC in lymph nodes represent a distinct subpopulation of DC that in this site are derived exclusively from
epidermal LC. Alternatively, TGF-
1 may modulate cell
surface expression of certain DC antigens. The deficiency of epidermal LC in TGF-
1
/
mice reported here also
constitutes one of the most striking abnormalities in hematopoetic cells that has been documented in these animals. Previous studies focused primarily on abnormalities in
the representation of mononuclear cell subpopulations in
lymphoid tissues, and the immune dysregulation that underlies the inflammatory wasting syndrome that is associated with this genotype (6, 7, 24). The relationship of the LC/DC abnormalities in TGF-
1
/
mice to other features of the phenotype is unknown, although abnormalities
in DC may contribute to the decreased T cell mitogenic responses that are seen in TGF-
1
/
mononuclear cells (24).
The nature of the requirement for TGF-1 as a regulator of LC has not been determined. TGF-
1 may be required for proliferation or differentiation of LC precursors.
This suggestion is compatible with the recent report that
TGF-
1 promotes the in vitro expansion of DC in serumfree cultures of human CD34+ hematopoetic progenitors
(25). Alternatively, TGF-
1 may influence the ability of
LC precursors (or LC) to localize in epidermis or facilitate
LC survival. Candidate mechanisms for TGF-
1 action include modulation of cytokine production (3), induction of growth factors or growth factor receptors (26), and regulation of adhesion molecule expression or function (27).
Regulation could occur at transcriptional or posttranscriptional levels. Interestingly, there are a number of similarities
between TGF-
1 null mice and mice that are deficient in
the transcription factor relB (28, 29). Inflammatory syndromes and DC abnormalites are present in both models.
RelB
/
mice have LC, but lack lymphoid DC. In contrast, TGF-
1
/
mice have lymphoid DC, but lack LC. These results, in light of the recent determination that
TGF-
1 influences the activity of NF-
B/Rel proteins in
murine B cells by regulating levels of the inhibitor I
B
(30), suggest that TGF-
1 may regulate DC via a similar
pathway. Resolution of these questions should result in
elucidation of mechanisms by which murine LC/DC are
regulated in vivo.
Address correspondence to Dr. Mark C. Udey, Dermatology Branch, National Cancer Institute, National Institutes of Health, Building 10, Room 12N238, Bethesda, MD 20892-1908.
Received for publication 28 August 1996
The authors thank Vivian McFarland for technical assistance, Harry Schaefer for help preparing the figures, Dr. Suren Sehgal (Wyeth-Ayerst) for supplying rapamycin, and Drs. George L. Barnes, Thilo Jakob, Stephen I. Katz, and Kim B. Yancey for reviewing the manuscript.
This work was supported in part by grants from the National Institutes of Health (AI 24137 and AG 04350 to A.G. Farr).
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