Immunology Today, vol. 5, No. 1, 1984
but not by soya bean trypsin inhibitor, suggesting that entrapped proteases, inhibitable only by the low molecular weight trasylol, are responsible for the activation. A. Gaspar (Chicago) and Teodorescu have devised a simple solidphase colourimetric assay for enzyme-a2-macroglobulin complexes which they call the polyclonal B-cell activator (PBA). PBA activity is raised in the serum of patients with rheumatoid arthritis and it was suggested that the generation of PBA in an inflammatory lesion might be an important stimulus for B-cell autoantibody production and the formation of immune complexes. One of the features of chronic inflammation is proliferation of the connective tissue cells and matrix. The process involves collagens, fibronectin, and other structural glycoproteins such as laminin. Typically, inflammation leads to changes in the amount and type of collagen produced: the rheumatoid synovium contains relatively greater amounts of collagen type III and changes in collagen may follow from activation of connective tissue cells by IL-1 or other mediators (C. Black, London). The interrelationships of one connective tissue protein, fibronectin, with mesenchymal cells,
Mechanisms in chronic inflammation from John Whicher and Robin Chambers A meeting was held recently to bring together ideas from workers in several different fields relating to chronic inflammation in rheumatology*. The protagonists were asked to explore four major themes: the acute-phase response; the control of lymphocyte function; the nature of the connective tissue matrix; and the role of iron in inflammation. The acute-phase response was considered in the broadest context as a series of events, mediated by the macrophagederived monokine interleukin 1 (IL-1), with differing functions in inflammation. The response encompasses the increase in serum acute-phase proteins, fever, leucocytosis, B-cell activation, interleukin 2 (IL-2) release, muscle proteolysis, and the activation of tissue cells such as chondrocytes, fibroblasts and synoviocytes. The recent finding that synovial factor (SF) and catabolin may be closely related to IL-1 suggests that cells other than the macrophage may be able to elicit these responses. There is interesting evidence that several acutephase proteins may be deposited on newly formed elastic fibres in healing wounds, suggesting a role for the proteins in the repair and resolution following inflammation. The notable absence of an acute-phase protein response to the disease process in a number of conditions, in particular systemic lupus erythematosus (M. Pepys and I. Rowe, London), and the failure of patients with systemic sclerosis to mount an acutephase response to prostaglandins (J. Whicher, Bristol) raise the question of whether defects in the response may be involved in some forms of inflammation. Why do lymphocytes migrate into an inflamed joint space? Are they responding to foreign or altered self antigens or are there other, non-antigenic factors? There is an accumulation of iron in the inflamed joint, as both .ferritin and haemosiderin,which correlates with the numbers oflymphocytes present. In sites of inflammation, polymorphs release lactoferrin and macrophages release apoferritin. Lactoferrin has a binding affinity for iron that is several hundred times greater than that of transferrin for iron, so that a shift of iron onto lactoferrin may explain the precipitous fall in serum iron seen in acute inflammation. Lymphocytes have membrane receptors for ferritin, lactoferrin and transferrinbound iron and zinc. It is possible that iron-binding proteins and iron-protein receptors may influence lymphoid cell *This meeting, organized by Paul Bacon and David Scott and colleagues, was held at the Universityof BirminghamMedical School, UK, on 8-9 September1983.
migration and lymphocyte activation (M. De Souza, New York). O f considerable interest is the observation that phytohaemagglutinin (PHA)-stimulated secretion of apoferritin by macrophages in H L A A~ individuals is defective, suggesting regulation by the major histocompatibility complex (MHC). M. Teodorescu (Chicago) discussed the possible role of a2-macroglobulinprotease complexes in B-cell activation and lymphocyte homing. This protease inhibitor is synthesized by hepatocytes and macrophages and is present in autologous mixed lymphocyte reaction (MLR) supernatants. The M L R derived a2-macrogtobulin acts as a B-cell activator when removed from a dialysable low molecular weight inhibitor and is probably derived from lymphocytes. B-cell activation is blocked by trasylol
?~~.x
CONNECTIVE TISSUEPROLIFERATIONII AND CELLULARACTIVATION
I
PROTEIN[ 1ACUTE-PHASE SYNTHES,S f', L-1 tL-1 [ pr.otease~ inhibition ~
"
,nd, to a fmacroglobutin IL-1 IPolyclonal TSt,mu,at,on/~'~ ~'acBt'iCvelt/r inhib~tion~~~,~
\ ~,.~ ~ _~ ,J "~~///~'~
[ ~roteases~ ~ ~ D~er-nrage t° ~
Altered I t ..... '1 I lrad~eeals
~"~----~---~ Fe
/4'ReceptOrs
ferritinorlactoferrin
Fig. 1. Some postulated.interactlonsbetween macrophages, iron, acute-phaseproteins
and lymphocytes. © 1984,ElsevxerSciencePubhshe~B.V.,Amsterdam 0167- 4919/84/$02.00
Fortechnicalreasonsweare unableto reproducethesefiguresin colourin thisedition--seetheJanuaryissueof ImmunologyTodayforfullcolourillustration.
Immunology Today, vol. 5, No. 1, 1984
fibrin and collagens were emphasized and it is possible that the interactions of this structural glycoprotein make it an important component of the chronic inflammatory process. In the rheumatoid joint, fibronectin complexes with hyaluronic acid and this complex lines the synovial cavity, influencing the interaction of many synovial fluid constituents with the underlying synovial cells. Also, collagen may be protected from proteolysis by complexing with locally synthesized fibronectin (I. Clemmensen, Copenhagen). Damage induced by free radicals may be of considerable importance in inflammation. B. Halliwell (London) outlined the pathways for the formation of O2-, O H - and H202. The evidence for direct inflammatory damage by O 2- and H202 is not impressive and suggests that the L y m p h o c y t e surface molecules - -
Contd. from#
1
domains furthest from the membrane defined the serology of HLA class-I antigens. Antigen receptors on T cells Perhaps the most exciting news of the week came with the progress reports on the characterization of a protein on the surface of T cells which may be responsible for recognition of antigen. A molecule which fulfils this role evidently must exist but its identity has remained strangely elusive until recently. Six groups have now reported 'anti-clonotypic' antibodies, which react selectively with various single clones of T cells and recognize putative receptor proteins of similar molecular weight. At this meeting M. Bevan (La Jolla) described two monoclonal antibodies raised against a mouse cytotoxic T-cell clone. Both selectively block killing by this clone and induce release of ]c-interferon. They precipitate an 80 000 tool. wt protein from the T cells which migrates as a 42 000 mol. wt band under reducing conditions. E. Reinherz (Boston) described anti-clonotypic antibodies against either a T4 + or a T8 ÷ human cytotoxic T-cell clone from the same individual, directed against a transformed B cell. They can selectively inhibit killing by each clone and block antigen stimulation of cell proliferation, though when conjugated to 'Sepharose' beads, they can induce proliferation of the appropriate clone. The protein structure of the antigens recognized by the anti-clonotypic antibodies is very similar to that reported by Bevan in the mouse. A 90 000 mol. wt protein is precipitated from both clones by the respective antibody. The human protein is resolved into two sub-units of
O2-derived product, O H - , though difficult to detect, may be more important. The most likely mechanism for its formation is by the reduction of iron salts. Certainly chelation of iron by desferrioxamine, but not EDTA, effectively inhibits the Haber-Weiss reaction forming O H - in iron-containing in-vitro systems. However, iron is not available in sufficient quantities in plasma, except in iron overload, but it is in synovial fluid and cerebrospinal fluid where /amol/1 quantities are present. The development of an assay for free iron using irondependent bleomycin-induced damage to DNA (D. Rowley, London) suggests that iron-binding proteins such as ferritin and lactoferrin in synovial fluid in arthritis are not binding iron as they should, and so enough free iron is available for O H - production. This may
John Whither and Robin Chambers are in the Department of Chemical Pathology, Bristol Royal Infirmary, Bristol BS2 8HW, UK.
tool. wt 49 000/51 000 and 43 000 on reducing gels, which isoelectric focusing and peptide mapping demonstrate are non-identical. Reinherz also discussed an intriguing connection between this putative T-cell receptor and the T3 antigen described earlier in the week by M. Crumpton (London). T3 is a marker o f T cells comprising a family of proteins of molecular weights ranging from 19 000 to 26 000. It is thought to be associated with antigen stimulation of lymphocyte proliferation, since anti-T3 antibodies act as polyclonal mitogens for resting human blood lymphocytes. Treatment of the human T-cell clones with anti-T3 antibodies cause a loss of surface expression of T3 and a comodulation of the clonotypic antigen. Anti-T3 antibodies can co-precipitate the characteristic 90 000 tool. wt protein. It therefore appears that the two structures are associated on the cell surface. Further circumstantial evidence in support of this came from the work of P. Beverley (London) who has isolated human antigen-specific helper T-cell clones. Prolonged exposure of these cells to antigen suppresses their subsequent helper function and proliferative response on re-stimulation by antigen. These 'tolerized' cells show reduced expression of T3 and anti-T3 antibodies can induce the same desensitized state. It seems therefore that with this new handle on a possible T-cell receptor at the protein level, another field as fertile for the molecular immunologist as the Ig system has prove d to be is now opening. In a very elegant talk K. Smith (Hanover, NH) discussed the factors which control the response of activated T cells to T cell growth factor (TCGF, or IL2). The T C G F receptor has been
identified as a 60 000 tool. wt species by cross-linking labelled T C G F or precipitation with an anti-TCGF receptor antibody and is only present on T cells activated by antigen or polyclonal mitogen. Fdllowing activation, the receptors appear asynchronously and are transiently expressed; analysis by fluorescence-activated cell sorter (FACS) therefore demonstrates that the receptor distribution on a population of activated cells is heterogeneous. When activated T cells are stimulated by TGGF there is a variable lag time before the cells start to synthesize DNA. FAGS analysis shows that the T ceils which have a longer lag time express lower levels of receptor. Smith has thus swept away a considerable amount of mystique which has surrounded previous observations of this sort in other cell types. Receptor concentration as well as ligand concentration can control response. The transferrin receptor has a ubiquitous distribution on the surface of proliferative cells, including T and B cells. M. Greaves (London) described the structure of the human transferrin receptor and discussed the possibility that it may be part of a large family of proteins involved in iron transport, all located on chromosome 3. This family may include the transferrin receptor, transferrin, lactoferrin and p97, a melanoma-associated antigen which binds iron and has sequence homology to transferrin. Rearrangements in this region of chromosome 3 may be associated with malignant transformation. I. Trowbridge (La Jolla) discussed the use of monoclonal antibodies to the transferrin receptor in the diagnosis and treatment of cancer and described interesting data concerning the use of two different
itself be due to free-radical-induced damage to the binding proteins (D. Blake, Birmingham). The possibility of manipulating chronic inflammation, apart from by well-tried methods such as prostaglandin synthesis, was the main theme of the meeting. The roles played by iron are still speculative, but when viewed together with new ideas about IL-1 further exciting aspects of inflammation become apparent. These are summarized in Fig. 1, which is a modified version of a figure presented at the meeting by P. Bacon (Birmingham). It is hoped that this will be the first in a series of meetings based on a similar theme.
but not by soya bean trypsin inhibitor, suggesting that entrapped proteases, inhibitable only by the low molecular weight trasylol, are responsible for the activation. A. Gaspar (Chicago) and Teodorescu have devised a simple solidphase colourimetric assay for enzyme-a2-macroglobulin complexes which they call the polyclonal B-cell activator (PBA). PBA activity is raised in the serum of patients with rheumatoid arthritis and it was suggested that the generation of PBA in an inflammatory lesion might be an important stimulus for B-cell autoantibody production and the formation of immune complexes. One of the features of chronic inflammation is proliferation of the connective tissue cells and matrix. The process involves collagens, fibronectin, and other structural glycoproteins such as laminin. Typically, inflammation leads to changes in the amount and type of collagen produced: the rheumatoid synovium contains relatively greater amounts of collagen type III and changes in collagen may follow from activation of connective tissue cells by IL-1 or other mediators (C. Black, London). The interrelationships of one connective tissue protein, fibronectin, with mesenchymal cells,
Mechanisms in chronic inflammation from John Whicher and Robin Chambers A meeting was held recently to bring together ideas from workers in several different fields relating to chronic inflammation in rheumatology*. The protagonists were asked to explore four major themes: the acute-phase response; the control of lymphocyte function; the nature of the connective tissue matrix; and the role of iron in inflammation. The acute-phase response was considered in the broadest context as a series of events, mediated by the macrophagederived monokine interleukin 1 (IL-1), with differing functions in inflammation. The response encompasses the increase in serum acute-phase proteins, fever, leucocytosis, B-cell activation, interleukin 2 (IL-2) release, muscle proteolysis, and the activation of tissue cells such as chondrocytes, fibroblasts and synoviocytes. The recent finding that synovial factor (SF) and catabolin may be closely related to IL-1 suggests that cells other than the macrophage may be able to elicit these responses. There is interesting evidence that several acutephase proteins may be deposited on newly formed elastic fibres in healing wounds, suggesting a role for the proteins in the repair and resolution following inflammation. The notable absence of an acute-phase protein response to the disease process in a number of conditions, in particular systemic lupus erythematosus (M. Pepys and I. Rowe, London), and the failure of patients with systemic sclerosis to mount an acutephase response to prostaglandins (J. Whicher, Bristol) raise the question of whether defects in the response may be involved in some forms of inflammation. Why do lymphocytes migrate into an inflamed joint space? Are they responding to foreign or altered self antigens or are there other, non-antigenic factors? There is an accumulation of iron in the inflamed joint, as both .ferritin and haemosiderin,which correlates with the numbers oflymphocytes present. In sites of inflammation, polymorphs release lactoferrin and macrophages release apoferritin. Lactoferrin has a binding affinity for iron that is several hundred times greater than that of transferrin for iron, so that a shift of iron onto lactoferrin may explain the precipitous fall in serum iron seen in acute inflammation. Lymphocytes have membrane receptors for ferritin, lactoferrin and transferrinbound iron and zinc. It is possible that iron-binding proteins and iron-protein receptors may influence lymphoid cell *This meeting, organized by Paul Bacon and David Scott and colleagues, was held at the Universityof BirminghamMedical School, UK, on 8-9 September1983.
migration and lymphocyte activation (M. De Souza, New York). O f considerable interest is the observation that phytohaemagglutinin (PHA)-stimulated secretion of apoferritin by macrophages in H L A A~ individuals is defective, suggesting regulation by the major histocompatibility complex (MHC). M. Teodorescu (Chicago) discussed the possible role of a2-macroglobulinprotease complexes in B-cell activation and lymphocyte homing. This protease inhibitor is synthesized by hepatocytes and macrophages and is present in autologous mixed lymphocyte reaction (MLR) supernatants. The M L R derived a2-macrogtobulin acts as a B-cell activator when removed from a dialysable low molecular weight inhibitor and is probably derived from lymphocytes. B-cell activation is blocked by trasylol
?~~.x
CONNECTIVE TISSUEPROLIFERATIONII AND CELLULARACTIVATION
I
PROTEIN[ 1ACUTE-PHASE SYNTHES,S f', L-1 tL-1 [ pr.otease~ inhibition ~
"
,nd, to a fmacroglobutin IL-1 IPolyclonal TSt,mu,at,on/~'~ ~'acBt'iCvelt/r inhib~tion~~~,~
\ ~,.~ ~ _~ ,J "~~///~'~
[ ~roteases~ ~ ~ D~er-nrage t° ~
Altered I t ..... '1 I lrad~eeals
~"~----~---~ Fe
/4'ReceptOrs
ferritinorlactoferrin
Fig. 1. Some postulated.interactlonsbetween macrophages, iron, acute-phaseproteins
and lymphocytes. © 1984,ElsevxerSciencePubhshe~B.V.,Amsterdam 0167- 4919/84/$02.00
Fortechnicalreasonsweare unableto reproducethesefiguresin colourin thisedition--seetheJanuaryissueof ImmunologyTodayforfullcolourillustration.
Immunology Today, vol. 5, No. 1, 1984
fibrin and collagens were emphasized and it is possible that the interactions of this structural glycoprotein make it an important component of the chronic inflammatory process. In the rheumatoid joint, fibronectin complexes with hyaluronic acid and this complex lines the synovial cavity, influencing the interaction of many synovial fluid constituents with the underlying synovial cells. Also, collagen may be protected from proteolysis by complexing with locally synthesized fibronectin (I. Clemmensen, Copenhagen). Damage induced by free radicals may be of considerable importance in inflammation. B. Halliwell (London) outlined the pathways for the formation of O2-, O H - and H202. The evidence for direct inflammatory damage by O 2- and H202 is not impressive and suggests that the L y m p h o c y t e surface molecules - -
Contd. from#
1
domains furthest from the membrane defined the serology of HLA class-I antigens. Antigen receptors on T cells Perhaps the most exciting news of the week came with the progress reports on the characterization of a protein on the surface of T cells which may be responsible for recognition of antigen. A molecule which fulfils this role evidently must exist but its identity has remained strangely elusive until recently. Six groups have now reported 'anti-clonotypic' antibodies, which react selectively with various single clones of T cells and recognize putative receptor proteins of similar molecular weight. At this meeting M. Bevan (La Jolla) described two monoclonal antibodies raised against a mouse cytotoxic T-cell clone. Both selectively block killing by this clone and induce release of ]c-interferon. They precipitate an 80 000 tool. wt protein from the T cells which migrates as a 42 000 mol. wt band under reducing conditions. E. Reinherz (Boston) described anti-clonotypic antibodies against either a T4 + or a T8 ÷ human cytotoxic T-cell clone from the same individual, directed against a transformed B cell. They can selectively inhibit killing by each clone and block antigen stimulation of cell proliferation, though when conjugated to 'Sepharose' beads, they can induce proliferation of the appropriate clone. The protein structure of the antigens recognized by the anti-clonotypic antibodies is very similar to that reported by Bevan in the mouse. A 90 000 mol. wt protein is precipitated from both clones by the respective antibody. The human protein is resolved into two sub-units of
O2-derived product, O H - , though difficult to detect, may be more important. The most likely mechanism for its formation is by the reduction of iron salts. Certainly chelation of iron by desferrioxamine, but not EDTA, effectively inhibits the Haber-Weiss reaction forming O H - in iron-containing in-vitro systems. However, iron is not available in sufficient quantities in plasma, except in iron overload, but it is in synovial fluid and cerebrospinal fluid where /amol/1 quantities are present. The development of an assay for free iron using irondependent bleomycin-induced damage to DNA (D. Rowley, London) suggests that iron-binding proteins such as ferritin and lactoferrin in synovial fluid in arthritis are not binding iron as they should, and so enough free iron is available for O H - production. This may
John Whither and Robin Chambers are in the Department of Chemical Pathology, Bristol Royal Infirmary, Bristol BS2 8HW, UK.
tool. wt 49 000/51 000 and 43 000 on reducing gels, which isoelectric focusing and peptide mapping demonstrate are non-identical. Reinherz also discussed an intriguing connection between this putative T-cell receptor and the T3 antigen described earlier in the week by M. Crumpton (London). T3 is a marker o f T cells comprising a family of proteins of molecular weights ranging from 19 000 to 26 000. It is thought to be associated with antigen stimulation of lymphocyte proliferation, since anti-T3 antibodies act as polyclonal mitogens for resting human blood lymphocytes. Treatment of the human T-cell clones with anti-T3 antibodies cause a loss of surface expression of T3 and a comodulation of the clonotypic antigen. Anti-T3 antibodies can co-precipitate the characteristic 90 000 tool. wt protein. It therefore appears that the two structures are associated on the cell surface. Further circumstantial evidence in support of this came from the work of P. Beverley (London) who has isolated human antigen-specific helper T-cell clones. Prolonged exposure of these cells to antigen suppresses their subsequent helper function and proliferative response on re-stimulation by antigen. These 'tolerized' cells show reduced expression of T3 and anti-T3 antibodies can induce the same desensitized state. It seems therefore that with this new handle on a possible T-cell receptor at the protein level, another field as fertile for the molecular immunologist as the Ig system has prove d to be is now opening. In a very elegant talk K. Smith (Hanover, NH) discussed the factors which control the response of activated T cells to T cell growth factor (TCGF, or IL2). The T C G F receptor has been
identified as a 60 000 tool. wt species by cross-linking labelled T C G F or precipitation with an anti-TCGF receptor antibody and is only present on T cells activated by antigen or polyclonal mitogen. Fdllowing activation, the receptors appear asynchronously and are transiently expressed; analysis by fluorescence-activated cell sorter (FACS) therefore demonstrates that the receptor distribution on a population of activated cells is heterogeneous. When activated T cells are stimulated by TGGF there is a variable lag time before the cells start to synthesize DNA. FAGS analysis shows that the T ceils which have a longer lag time express lower levels of receptor. Smith has thus swept away a considerable amount of mystique which has surrounded previous observations of this sort in other cell types. Receptor concentration as well as ligand concentration can control response. The transferrin receptor has a ubiquitous distribution on the surface of proliferative cells, including T and B cells. M. Greaves (London) described the structure of the human transferrin receptor and discussed the possibility that it may be part of a large family of proteins involved in iron transport, all located on chromosome 3. This family may include the transferrin receptor, transferrin, lactoferrin and p97, a melanoma-associated antigen which binds iron and has sequence homology to transferrin. Rearrangements in this region of chromosome 3 may be associated with malignant transformation. I. Trowbridge (La Jolla) discussed the use of monoclonal antibodies to the transferrin receptor in the diagnosis and treatment of cancer and described interesting data concerning the use of two different
itself be due to free-radical-induced damage to the binding proteins (D. Blake, Birmingham). The possibility of manipulating chronic inflammation, apart from by well-tried methods such as prostaglandin synthesis, was the main theme of the meeting. The roles played by iron are still speculative, but when viewed together with new ideas about IL-1 further exciting aspects of inflammation become apparent. These are summarized in Fig. 1, which is a modified version of a figure presented at the meeting by P. Bacon (Birmingham). It is hoped that this will be the first in a series of meetings based on a similar theme.
