(:LINICAI.
IMMUNOLOGY
Ii Blood
AND
IMMUNOPATHOLOGY
Group
Antigens
B. H. ToH,* Department
of Pathology
12, 177-182 (1979)
on Fibroblast
T. A. DIGGLE,
and Immunology, Melbourne, Victoria
Cell Surfaces’
AND S. H. KOH
Monash University 3181. Australia
Medical
School,
Received June 6. 1978 Six cold agglutinin-containing sera, directed against I or i antigens, gave immunofluorescent staining of stromal cells in frozen sections of tissues in vivo and in monolayer cultures of fibroblasts in vitro. Microcytotoxicity tests showed that the cold agglutinins were cytotoxic to Iibroblasts. The immunofluorescent staining probably represents serum reactivity with Ii antigens on tibroblast cell surfaces because the reactions were neutralized by serum absorption with fibroblasts or red blood cells, but not by absorption with hepatocytes. The sites on fibroblasts binding to cold agglutinins were resistant to tryptic digestion and remained on the glass substratum after cell detachment induced by trypsin.
INTRODUCTION
It has been suggested that the cell membrane may have important roles in cell recognition, communication, adhesion, division, and motility (1, 2). A number of surface components have been defined on fibroblasts, the cell type commonly used in studies on cultured cells. These include fibronectin, a cell-surface glycoprotein present in normal and lost in neoplastic fibroblasts (3). More recently, a collagen-like protein has also been demonstrated on fibroblast cell surfaces and it was suggested that this protein, together with fibronectin, may participate in cell interaction with the extracellular matrix (4). During routine screening of human sera for autoantibodies, we observed bright immunofluorescent staining of cords of cells in the kidney medulla with a serum containing cold agglutinins. Further studies with cold agglutinin-containing sera, designated anti-I, anti-adult red blood cells (RBC) or anti-i, anti-cord RBC (5), showed that the kidney medullary staining represented reactivity with Ii blood group antigens on fibroblast cell surfaces and that cell-bound cold agglutinins, in the presence of complement, were cytotoxic to fibroblasts it1 vim. The observation that the antigen persists in sites of cells detached by trypsin suggests that it may have a role in cell attachment to the underlying substratum. MATERIALS
AND METHODS
Zmmunofluorescent staining. Frozen sections of rat, mouse? rabbit, or chicken tissues and viable monolayer cultures of rat, human, or chicken fibroblasts were examined for immunofluorescence reactivity (6, 7) at 4”C, room temperature (15-209, or 37°C with 23 sera containing cold agglutinins. Control experiments for fibroblast surface staining were carried out by testing viable fibroblast
I Supported by grants from the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. z To whom requests for reprints should be sent. 177 0090-1229/79/020177-06$01.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
178
TOH,
DIGGLE,
AND
KOH
FIG. 1. Indirect (sandwich) immunofluorescent staining of frozen sections of tissues or monolayer cultures of fibroblasts by human sera containing cold agglutinins. (a) Rat kidney section reacted with anti-1 (Ken). showing staining of cords of interstitial cells in the medulla. x 200. Cb) Higher power of
CANTIGENS
0~
FIBROBLA~T
CELL
SURFACES
179
FIG. l--Continued. similar field as in (a), showing fine medulla. x 500. (c) Rat ileum section, cells; mucosal epithelial cells did not anti-1 (Ken) showing fine particulate that staining pattern is similar to that
particulate staining of spindle-shaped interstitial cells in the reacted with anti-1 (Ken), showing staining of elongated stromal stain. x 320. (d) Rat fetal tibroblast monolayer, reacted with staining distributed uniformly over the entire cell surface. Note in (b). x 500.
180
I-OH.
DIGGLE,
:\NL)
KOH
monolayers with autoantibodies directed against nuclei, smooth muscle, gastric parietal cells, mitochondria, kidney brush border. and thyroid cytoplasm or colloid (6). Fibroblast monolayers were also examined after treatment with 0.0015% trypsin at 37°C for S to 60 min (7). Any bound immunoglobulin was traced with a fluorescein-isothiocyanate (FITC)-labeled goat anti-human y-globulin or with monospecific FITC-labeled anti-IgM, IgG, or IgA (8). Immunoabsorption studies were carried out by mixing 0.2 ml of a 1:8 dilution of serum with an equal volume of packed fibroblasts, adult RBC. or cord RBC and incubating overnight with continuous agitation at 4°C. The supernatants were recovered by centrifugation at 2SOg for 10 min. Eluates were obtained by incubating the centrifuged pellets with 0.2 ml of phosphate-buffered saline (PBS) at 37°C. Control absorptions were carried out with rat hepatocytes obtained by perfusion of rat liver with 10 ml of calcium- and magnesium-free Hanks’ balanced salt solution containing 0.05% collagenase and 0.1% hyaluronidase and incubation of the diced tissue in the enzyme mixture at 37°C for 30 min. The absorbed sera and eluates were examined for immunofluorescent reactivity with frozen sections of tissues and with fibroblast monolayers. Microcytotoxicity tests. These were carried out by modification of the method of Bloom (9). Wells in microtitration plates were seeded with 10 ~1 of IO4 fibroblastsiml TC medium 199 (Ml99) containing 10% fetal calf serum. The plates were incubated at 37°C in 5% CO,: 955% air for 24 hr. After four washes in M 199, S ~1 of anti-1 (Ken) or normal human serum was added to five replicate wells in dilutions increasing from 1:8 to 1:256. The plates were left for 30 min at 4 or 37°C and the cells were treated with S ~1 of 1: 10 dilution guinea pig complement for S hr at room temperature ( IS-20°C). The cells were washed four times with PBS, fixed in absolute methanol, and counted. Percentage cytotoxicity of tests at 4 or 37°C was expressed as [(NC-Nt)/Nc] x 100 where NC is the mean cell number in wells containing normal human serum and Nt is the mean cell number in wells containing anti-1 (Ken). RESULTS
AND DISCUSSION
Six out of twenty-three cold agglutinin sera gave immunofluorescent staining of cords of interstitial cells in the medulla offrozen sections of rat kidney (Fig. la). The six sera comprised anti-1 (Ken) from a patient with idiopathic cold agglutinin syndrome, anti-1 (Arm) and anti-1 (Sch) from patients with M~coplasm~~ przeumoniue infections, anti-i (Tas) from a patient with infectious mononucleosis, and anti-1 (Craw) and anti-i (Grin) from patients in whom no diagnosis was established (Table 1). Unfortunately, the sera were not examined for various Pr specificities. It is thus conveivable that of the 17 sera nonreactive by immunofluorescence, all may have been anti-Prl, anti-Pr2, etc. In addition, some of the “anti-I” may have been anti-Pr. Higher magnification showed that staining in a fine particulate pattern was present in spindle-shaped cells (Fig. lb). Longitudinal and transverse sections of rat esophagus, stomach, and small and large intestine also showed staining of spindle-shaped stromal cells; parenchymal cells did not stain (Fig. lc). Thymus, lymph nodes, and spleen showed staining of connective tissue septa while lymphocytes did not stain. Ovary, fallopian tubes, and uterus showed
ri
ANTIGENS
IMMUNOFLUORESCENT (15520°C)
0~
FIBROBLAST
crm.
TABLE 1 STAINING OF FIBROBLASTS AT 4°C OR ROOM BY SERA COYI-AISIN~ COLD AG~L~TININS” Hemagglutination Human adult RBC
Cold agglutinins Anti-I Anti-I Anti-I Anti-I Anti-i Anti-i 0 Optimal 37°C.
(Arm) (Ken) (Craw) (Sch) (Tas) (Grin)
staining
was seen at 4°C and weaker
or immunofluorescent Human cord RBC
5096 1024 1024 512 64 64
181
SURFACES
TEMPERATURE
staining
Rat tibroblasts
1024 128 8 8 256 256 staining
at room
titer
64 128 128 64 256 64
temperature.
Staining
was absent
at
staining restricted to serosal lining cells. Brain, heart, and skeletal muscle did not stain. Mouse, rabbit, and chicken tissue gave similar staining patterns. Viable or fixed monolayer cultures of fetal or embryonic rat fibroblasts showed a staining pattern similar to that seen in tissue sections (Fig. Id). Identical patterns of staining were seen in sparse or confluent cultures and in cultures of embryonic human or chicken tibroblasts. Staining was not inhibited by treating viable cells with 0.001% trypsin at 37°C for 5 to 60 min. The sites of trypsin-detached cells were marked by positive-staining fine aggregates. These may represent “footpads” (lo), points of cell attachment to the glass substratum. Fibroblast surface staining was not seen in viable cultured cells reacted with autoantibodies directed against nuclei, smooth muscle, gastric parietal cells, mitochondria, kidney brush borders, and thyroid cytoplasm or colloid. Staining of tissue sections and fibroblast monolayers was seen at 4°C or room temperature (15 -20°C) but not at 37”C, and with FITC-labeled anti-human y-globulin or anti-human IgM but not with anti-IgG or anti-IgA conjugates. Immunoabsorption studies with anti-1 (Ken) and anti-i (Grin) showed that fibroblast staining and hemagglutination was inhibited by serum absorption with TABLE 2 H~MAGGLUTISATION OR IMMUSOFLUORESCENT STAINISG TITERS OF ADULT RBC, FIBROBLASTS, AND HEPATO~I.~E.S OBT-AINED WITH COLD AGGLUTININ SERA DIRECTED AGAISST I OR i ANTIGENS Titers
obtained
Serum absorptions
Human adult RBC
Human cord RBC
Nil +AduIt RBC +Cord RBC +Fibroblasts +Hepatocytes
1024 18 128 32 1024
128
IMMUNOLOGY
Ii Blood
AND
IMMUNOPATHOLOGY
Group
Antigens
B. H. ToH,* Department
of Pathology
12, 177-182 (1979)
on Fibroblast
T. A. DIGGLE,
and Immunology, Melbourne, Victoria
Cell Surfaces’
AND S. H. KOH
Monash University 3181. Australia
Medical
School,
Received June 6. 1978 Six cold agglutinin-containing sera, directed against I or i antigens, gave immunofluorescent staining of stromal cells in frozen sections of tissues in vivo and in monolayer cultures of fibroblasts in vitro. Microcytotoxicity tests showed that the cold agglutinins were cytotoxic to Iibroblasts. The immunofluorescent staining probably represents serum reactivity with Ii antigens on tibroblast cell surfaces because the reactions were neutralized by serum absorption with fibroblasts or red blood cells, but not by absorption with hepatocytes. The sites on fibroblasts binding to cold agglutinins were resistant to tryptic digestion and remained on the glass substratum after cell detachment induced by trypsin.
INTRODUCTION
It has been suggested that the cell membrane may have important roles in cell recognition, communication, adhesion, division, and motility (1, 2). A number of surface components have been defined on fibroblasts, the cell type commonly used in studies on cultured cells. These include fibronectin, a cell-surface glycoprotein present in normal and lost in neoplastic fibroblasts (3). More recently, a collagen-like protein has also been demonstrated on fibroblast cell surfaces and it was suggested that this protein, together with fibronectin, may participate in cell interaction with the extracellular matrix (4). During routine screening of human sera for autoantibodies, we observed bright immunofluorescent staining of cords of cells in the kidney medulla with a serum containing cold agglutinins. Further studies with cold agglutinin-containing sera, designated anti-I, anti-adult red blood cells (RBC) or anti-i, anti-cord RBC (5), showed that the kidney medullary staining represented reactivity with Ii blood group antigens on fibroblast cell surfaces and that cell-bound cold agglutinins, in the presence of complement, were cytotoxic to fibroblasts it1 vim. The observation that the antigen persists in sites of cells detached by trypsin suggests that it may have a role in cell attachment to the underlying substratum. MATERIALS
AND METHODS
Zmmunofluorescent staining. Frozen sections of rat, mouse? rabbit, or chicken tissues and viable monolayer cultures of rat, human, or chicken fibroblasts were examined for immunofluorescence reactivity (6, 7) at 4”C, room temperature (15-209, or 37°C with 23 sera containing cold agglutinins. Control experiments for fibroblast surface staining were carried out by testing viable fibroblast
I Supported by grants from the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. z To whom requests for reprints should be sent. 177 0090-1229/79/020177-06$01.00/O Copyright All rights
6 1979 by Academic Press, Inc. of reproduction in any form reserved.
178
TOH,
DIGGLE,
AND
KOH
FIG. 1. Indirect (sandwich) immunofluorescent staining of frozen sections of tissues or monolayer cultures of fibroblasts by human sera containing cold agglutinins. (a) Rat kidney section reacted with anti-1 (Ken). showing staining of cords of interstitial cells in the medulla. x 200. Cb) Higher power of
CANTIGENS
0~
FIBROBLA~T
CELL
SURFACES
179
FIG. l--Continued. similar field as in (a), showing fine medulla. x 500. (c) Rat ileum section, cells; mucosal epithelial cells did not anti-1 (Ken) showing fine particulate that staining pattern is similar to that
particulate staining of spindle-shaped interstitial cells in the reacted with anti-1 (Ken), showing staining of elongated stromal stain. x 320. (d) Rat fetal tibroblast monolayer, reacted with staining distributed uniformly over the entire cell surface. Note in (b). x 500.
180
I-OH.
DIGGLE,
:\NL)
KOH
monolayers with autoantibodies directed against nuclei, smooth muscle, gastric parietal cells, mitochondria, kidney brush border. and thyroid cytoplasm or colloid (6). Fibroblast monolayers were also examined after treatment with 0.0015% trypsin at 37°C for S to 60 min (7). Any bound immunoglobulin was traced with a fluorescein-isothiocyanate (FITC)-labeled goat anti-human y-globulin or with monospecific FITC-labeled anti-IgM, IgG, or IgA (8). Immunoabsorption studies were carried out by mixing 0.2 ml of a 1:8 dilution of serum with an equal volume of packed fibroblasts, adult RBC. or cord RBC and incubating overnight with continuous agitation at 4°C. The supernatants were recovered by centrifugation at 2SOg for 10 min. Eluates were obtained by incubating the centrifuged pellets with 0.2 ml of phosphate-buffered saline (PBS) at 37°C. Control absorptions were carried out with rat hepatocytes obtained by perfusion of rat liver with 10 ml of calcium- and magnesium-free Hanks’ balanced salt solution containing 0.05% collagenase and 0.1% hyaluronidase and incubation of the diced tissue in the enzyme mixture at 37°C for 30 min. The absorbed sera and eluates were examined for immunofluorescent reactivity with frozen sections of tissues and with fibroblast monolayers. Microcytotoxicity tests. These were carried out by modification of the method of Bloom (9). Wells in microtitration plates were seeded with 10 ~1 of IO4 fibroblastsiml TC medium 199 (Ml99) containing 10% fetal calf serum. The plates were incubated at 37°C in 5% CO,: 955% air for 24 hr. After four washes in M 199, S ~1 of anti-1 (Ken) or normal human serum was added to five replicate wells in dilutions increasing from 1:8 to 1:256. The plates were left for 30 min at 4 or 37°C and the cells were treated with S ~1 of 1: 10 dilution guinea pig complement for S hr at room temperature ( IS-20°C). The cells were washed four times with PBS, fixed in absolute methanol, and counted. Percentage cytotoxicity of tests at 4 or 37°C was expressed as [(NC-Nt)/Nc] x 100 where NC is the mean cell number in wells containing normal human serum and Nt is the mean cell number in wells containing anti-1 (Ken). RESULTS
AND DISCUSSION
Six out of twenty-three cold agglutinin sera gave immunofluorescent staining of cords of interstitial cells in the medulla offrozen sections of rat kidney (Fig. la). The six sera comprised anti-1 (Ken) from a patient with idiopathic cold agglutinin syndrome, anti-1 (Arm) and anti-1 (Sch) from patients with M~coplasm~~ przeumoniue infections, anti-i (Tas) from a patient with infectious mononucleosis, and anti-1 (Craw) and anti-i (Grin) from patients in whom no diagnosis was established (Table 1). Unfortunately, the sera were not examined for various Pr specificities. It is thus conveivable that of the 17 sera nonreactive by immunofluorescence, all may have been anti-Prl, anti-Pr2, etc. In addition, some of the “anti-I” may have been anti-Pr. Higher magnification showed that staining in a fine particulate pattern was present in spindle-shaped cells (Fig. lb). Longitudinal and transverse sections of rat esophagus, stomach, and small and large intestine also showed staining of spindle-shaped stromal cells; parenchymal cells did not stain (Fig. lc). Thymus, lymph nodes, and spleen showed staining of connective tissue septa while lymphocytes did not stain. Ovary, fallopian tubes, and uterus showed
ri
ANTIGENS
IMMUNOFLUORESCENT (15520°C)
0~
FIBROBLAST
crm.
TABLE 1 STAINING OF FIBROBLASTS AT 4°C OR ROOM BY SERA COYI-AISIN~ COLD AG~L~TININS” Hemagglutination Human adult RBC
Cold agglutinins Anti-I Anti-I Anti-I Anti-I Anti-i Anti-i 0 Optimal 37°C.
(Arm) (Ken) (Craw) (Sch) (Tas) (Grin)
staining
was seen at 4°C and weaker
or immunofluorescent Human cord RBC
5096 1024 1024 512 64 64
181
SURFACES
TEMPERATURE
staining
Rat tibroblasts
1024 128 8 8 256 256 staining
at room
titer
64 128 128 64 256 64
temperature.
Staining
was absent
at
staining restricted to serosal lining cells. Brain, heart, and skeletal muscle did not stain. Mouse, rabbit, and chicken tissue gave similar staining patterns. Viable or fixed monolayer cultures of fetal or embryonic rat fibroblasts showed a staining pattern similar to that seen in tissue sections (Fig. Id). Identical patterns of staining were seen in sparse or confluent cultures and in cultures of embryonic human or chicken tibroblasts. Staining was not inhibited by treating viable cells with 0.001% trypsin at 37°C for 5 to 60 min. The sites of trypsin-detached cells were marked by positive-staining fine aggregates. These may represent “footpads” (lo), points of cell attachment to the glass substratum. Fibroblast surface staining was not seen in viable cultured cells reacted with autoantibodies directed against nuclei, smooth muscle, gastric parietal cells, mitochondria, kidney brush borders, and thyroid cytoplasm or colloid. Staining of tissue sections and fibroblast monolayers was seen at 4°C or room temperature (15 -20°C) but not at 37”C, and with FITC-labeled anti-human y-globulin or anti-human IgM but not with anti-IgG or anti-IgA conjugates. Immunoabsorption studies with anti-1 (Ken) and anti-i (Grin) showed that fibroblast staining and hemagglutination was inhibited by serum absorption with TABLE 2 H~MAGGLUTISATION OR IMMUSOFLUORESCENT STAINISG TITERS OF ADULT RBC, FIBROBLASTS, AND HEPATO~I.~E.S OBT-AINED WITH COLD AGGLUTININ SERA DIRECTED AGAISST I OR i ANTIGENS Titers
obtained
Serum absorptions
Human adult RBC
Human cord RBC
Nil +AduIt RBC +Cord RBC +Fibroblasts +Hepatocytes
1024 18 128 32 1024
128
