Production of B cell-stimulating factors by B cells in patients with systemic lupus erythematosus

The production of B cell-stimulating factors (BSF) by B cells in patients with systemic lupus erythematosus (SLE) was studied in vitro. B cells from SLE patients markedly proliferated and differentiated into Ig-producing cells by in vitro culture without any stimulation. The culture supernatant of these B cells contained BSF activity that stimulated Staphylococcus aureus Cowan I-treated normal B cells to proliferate and differentiate into Ig-producing cells. By a Percoll gradient density centrifugation, BSF-producing cells were enriched in the higher density fraction, but were reduced in the lower density fraction. The BSF also stimulated the proliferation and the differentiation of SLE B cells. By a Percoll gradient density centrifugation, SLE B cells responsive to the BSF were enriched in the higher density fraction, but were reduced in the lower density fraction. The Mr of the BSF was estimated as about 18,000 Da by Sephacryl S-200 column chromatography. The BSF fraction did not possess IL-2 and IFN activity, but possessed IL-1 activity, which stimulated murine thymocyte proliferative responses. The BSF activity was partially, but not completely, absorbed by an anti-IL-1 alpha antibody. Furthermore, the BSF possessed IL-4 activity, which induced not only the proliferative responses of normal B cells stimulated with B cell mitogens, but also the expression of low affinity Fc epsilon R/CD23 on normal B cells. The BSF also possessed IL-6 activity, which induced the proliferative responses of IL-6-dependent hybridoma cells, MH-60 BSF2. Moreover, human rIL-1, rIL-4, and rIL-6 stimulated SLE B cells. These results suggest that SLE B cells spontaneously produce the BSF such as IL-1 alpha, IL-4, and IL-6 and express their receptors on their surface, and the interaction between the BSF and their receptors stimulates SLE B cells to spontaneously proliferate and differentiate into Ig-producing cells as an autocrine mechanism.     

Detection of receptors for murine B cell stimulatory factor 1 (BSF1): presence of functional receptors on CBA/N splenic B cells

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.     

Stimulation of EBV-activated human B cells by monocytes and monocyte products. Role of IFN-beta 2/B cell stimulatory factor 2/IL-6

EBV infects human B lymphocytes and induces them to proliferate, to produce Ig, and to give rise to immortal cell lines. Although the mechanisms of B cell activation by EBV are largely unknown, the continuous proliferation of EBV-immortalized B cells is dependent, at least in part, upon autocrine growth factors produced by the same EBV-infected B cells. In the present studies we have examined the influence of monocytes on B cell activation by EBV and found that unlike peripheral blood T cells and B cells, monocytes enhance by as much as 30- to 50-fold virus-induced B cell proliferation and Ig production. Upon activation with LPS, monocytes secrete a growth factor activity that promotes both proliferation and Ig secretion in EBV-infected B cells and thus reproduces the effects of monocytes in these cultures. Unlike a number of other factors, rIFN-beta 2/B cell stimulatory factor 2 (BSF-2)/IL-6 stimulates the growth of human B cells activated by EBV in a manner similar to that induced by activated monocyte supernatants. In addition, an antiserum to IFN-beta that recognizes both IFN-beta 1 and IFN-beta 2 completely neutralizes the B cell growth factor activity of activated monocyte supernatants. These findings demonstrate that IFN-beta 2/BSF-2/IL-6 is a growth factor for human B cells activated by EBV and suggest that this molecule is responsible for B cell growth stimulation induced by activated monocyte supernatants. We have examined the possibility that IFN-beta 2/BSF-2/IL-6 might also be responsible for B cell growth stimulation by supernatants of EBV-immortalized B cells and thus may function as an autocrine growth factor. However, IFN-beta 2/BSF-2/IL-6 is not detectable in supernatants of EBV-immortalized B cells by immunoprecipitation. Also, an antiserum to IFN-beta that neutralizes IFN-beta 2/BSF-2/IL-6 fails to neutralize autocrine growth factor activity. This suggests that autocrine growth factors produced by EBV-immortalized B cells are distinct from IFN-beta 2/BSF-2/IL-6. Thus, the continuous proliferation of EBV-immortalized B cells is enhanced by either autocrine or paracrine growth factors. One of the mediators with paracrine growth factor activity is IFN-beta 2/BSF-2/IL-6.     

IFN-beta 2, B cell differentiation factor 2, or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines

IL-6, which is also known as IFN-beta 2, hybridoma growth factor, hepatocyte-stimulating factor, and B cell differentiation factor, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, fibroblasts, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term keratinocyte cultures, A431, or KB cells stimulated the proliferation of a hybridoma growth factor/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or fibroblast-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within 21 and 28 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta 2/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell growth-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured keratinocytes and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating factor 2/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular keratinocytes also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.     

Interleukin-6: discovery of a pleiotropic cytokine

In the late 1960s, the essential role played by T cells in antibody production was reported. This led to our hypothesis that certain molecules would have to be released from T cells to effect the stimulation of B cells. This hypothesis was shown to be true. There were certain factors present in the culture supernatant of T cells that induced proliferation and differentiation of B cells. The factor that induced B cells to produce immunoglobulins was initially named B cell stimulatory factor-2. The cDNA encoding the human B cell stimulatory factor-2 was cloned in 1986. At the same time, IFN-beta2 and a 26 kDa protein in the fibroblasts were independently cloned and found to be identical to B cell stimulatory factor-2. Later, a hybridoma/plasmacytoma growth factor and a hepatocyte stimulating factor were also proven to be the same molecule as B cell stimulatory factor-2. Various names were used for this single molecule because of its multiple biological activities, but these have all been unified and the molecule is now known as IL-6. Since the discovery of IL-6, rapid progress has been made in our understanding of IL-6 activities, the IL-6 receptor system and the IL-6 signal transduction mechanism. More importantly, it has been shown to be involved in a number of diseases such as rheumatoid arthritis and Castleman's disease. When taking into account all the accumulated basic research on the various aspects of this molecule, it appeared that blocking the activity of IL-6 was a feasible, new therapeutic approach for chronic inflammatory diseases.     

IL-1 and tumor necrosis factor. Similarities and differences in stimulation of expression of alternative pathway of complement and IFN-beta 2/IL-6 genes in human fibroblasts

IL-1 and TNF induced concentration-related increases in the synthesis of factor B, C3, and IFN-beta 2/IL-6 in human skin fibroblasts. Effects of both stimuli were apparent with concentrations as low as 0.1 ng/ml and maximal responses were observed between 1 and 10 ng/ml; only for IL-1 induction of IFN-beta 2/IL-6 was there a further increase in response up to 100 ng/ml. For factor B and C3, maximal increases induced by IL-1 and TNF were similar: 119- and 109-fold for factor B and 15-fold and 11-fold for C3, respectively. Although both IL-1 and TNF increase synthesis of factor B and C3 in hepatocytes, the increases observed in fibroblasts were approximately 50- and 8-fold more for factor B and C3, respectively. Neither protein synthesis nor mRNA for IFN-beta 2/IL-6 was present in HepG2 cells either before or after stimulation with IL-1 or TNF. In contrast to the similarities between the effects of IL-1 and TNF on synthesis of factor B, C3, and IFN-beta 2/IL-6, only TNF increased synthesis of factor H. Because TNF induces membrane IL-1 in fibroblasts, it is possible to speculate that the effects of TNF on fibroblasts are due to induction of IL-1. An autocrine action of TNF through IL-1 is possible for TNF-induced synthesis of IFN-beta 2/IL-6, but the effects of TNF on synthesis of factor B, C3, and factor H indicated that TNF has effects on fibroblasts separate from IL-1. The effects of IL-1 and TNF on the synthesis of factor B and C3 in fibroblasts may be a part of an acute phase response occurring at a local level. However, the large responses in synthesis of factor B and C3 to IL-1 and TNF may suggest that factor B and C3 have a role, as yet undescribed, in tissues in addition to the role these proteins are known to play in inflammation.     

Characterization of a keratinocyte-derived T cell growth factor distinct from interleukin 2 and B cell stimulatory factor 1

Epidermal epithelial cells (keratinocytes) produce and secrete a variety of immunologically active cytokines. We have previously reported that both transformed (PAM 212) and normal murine keratinocytes produce a soluble factor which induces proliferation of the T cell line, HT-2. In the present study we sought to compare keratinocyte-derived T cell growth factor (KTGF) with other T cell growth factors, characterize its physicochemical properties, and substantially purify KTGF from PAM 212 conditioned medium. KTGF from PAM 212 conditioned medium was not inhibited by antibodies which block the effect of interleukin 2 (IL 2) (S4B6) or B cell stimulatory factor 1 (BSF 1) (11B11). KTGF is heat-stable, has an isoelectric point of 4.8, and a relative molecular mass of 16 to 23 kilodaltons under nonreducing conditions. KTGF activity was enhanced at least 41,413-fold by sequential hydroxylapatite bulk preparation, desalting by reversed-phase chromatography, gel filtration high pressure liquid chromatography (HPLC), and reversed-phase HPLC. Keratinocytes produce a T cell growth factor with physicochemical properties distinct from IL 2 and BSF 1. KTGF may play a role in regulating the growth and differentiation of T cells in the epidermis.     

Human recombinant IL-3 is a growth factor for normal B cells.

IL-3 is a well known hemopoietic cell growth and differentiation factor. However, its functional role in normal B cell differentiation has not been established. We have investigated the effect of IL-3 on the growth and differentiation of human B cells. IL-3 enhanced the proliferation of Staphylococcus aureus Cowan 1 strain-stimulated B cells. The optimal time of IL-3 to stimulate B cell growth was on day 2 to day 3, suggesting that IL-3 was a B cell growth factor acting in the late stage. IL-3 synergized with IL-2 to enhance B cell proliferation and differentiation. Pretreatment of B cells with IL-3 for more than 3 days increased the expression of IL-2R on B cells. However, pretreatment of B cells with IL-2 did not alter the subsequent response to IL-3, suggesting that the synergy between IL-2 and IL-3 may be attributed to the up-regulation of IL-2 response by IL-3. In addition, pretreatment of B cells with IL-4 decreased subsequent response of B cells to IL-3 as well as IL-2, suggesting that IL-3- and IL-2-responding cells passed a similar way during the early stage of B cell activation. It appears that IL-3 and IL-6 mediate normal B cell differentiation via separate mechanisms. IL-3-induced B cell differentiation was mainly mediated by increasing cell growth, whereas IL-6 induced B cell differentiation without affecting proliferation.     

Physicochemical and functional properties of murine B cell-derived B cell growth factor II (WEHI-231-BCGF-II)

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

γ-Tocotrienol inhibits lipopolysaccharide-induced interlukin-6 and granulocyte colony-stimulating factor by suppressing C/EBPβ and NF-κB in macrophages

Cytokines generated from macrophages contribute to pathogenesis of inflammation-associated diseases. Here we show that γ-tocotrienol (γ-TE), a natural vitamin E form, inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-6 production without affecting tumor necrosis factor α (TNF-α), IL-10 or cyclooxygenase-2 (COX-2) up-regulation in murine RAW264.7 macrophages. Mechanistic studies indicate that nuclear factor κB (NF-κB), but not c-Jun NH(2)-terminal protein kinase, p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs), is important to IL-6 production and that γ-TE treatment blocks NF-κB activation. In contrast, COX-2 appears to be regulated by p38 MAPK in RAW cells, but γ-TE has no effect on LPS-stimulated p38 phosphorylation. Despite necessary for IL-6, NF-κB activation by TNF-α or other cytokines is not sufficient for IL-6 induction with exception of LPS. CCAAT/enhancer-binding protein (C/EBP) β appears to be involved in IL-6 formation because LPS induces C/EBPβ up-regulation, which parallels IL-6 production, and knockdown of C/EBPβ with small interfering RNA results in diminished IL-6. LPS but not individual cytokines is capable of stimulating C/EBPβ and IL-6 in macrophages. Consistent with its dampening effect on IL-6, γ-TE blunts LPS-induced up-regulation of C/EBPβ without affecting C/EBPδ. γ-TE also decreases LPS-stimulated granulocyte colony-stimulating factor (G-CSF), a C/EBPβ target gene. Compared with RAW264.7 cells, γ-TE shows similar or stronger inhibitory effects on LPS-triggered activation of NF-κB, C/EPBβ and C/EBPδ and more potently suppresses IL-6 and G-CSF in bone marrow-derived macrophages. Our study demonstrates that γ-TE has antiinflammatory activities by inhibition of NF-κB and C/EBPs activation in macrophages.     

Stimulation of murine hemopoietic colony formation by human IL-6

A novel hemopoietic CSF has been identified in the medium conditioned by lectin-stimulated human T cells. The cDNA clone encoding this factor, isolated by functional expression cloning in monkey cos-1 cells, proved to be identical with the cDNA encoding the cytokine B cell stimulatory factor-2/IFN-beta 2, a factor now known as IL-6. In the murine system, IL-6 indirectly supports the formation of several different types of hemopoietic colonies, including those derived from early blast cells, and directly supports the proliferation of granulocyte/macrophage progenitors. These results expand the range of known target cells of IL-6 to include hemopoietic progenitors in addition to B cells, T cells, and fibroblasts and provide further evidence that this cytokine plays an important role within a network of interacting cytokines that regulates many different biologic responses.     

Human recombinant IL-3 stimulates B cell differentiation

To investigate the role of human IL-3 in B cell differentiation, we examined its effect on IgG secretion from normal B cells and a B cell line, JDA. The effect of IL-3 was compared to that of IL-6. IL-3 stimulated IgG secretion from tonsil B cells or peripheral blood-derived B cells activated by Staphylococcus aureus Cowan I strain. This effect required the presence of IL-2. Neither B cell growth factor (BCGF) nor IFN-gamma replaced IL-2 in this function. IL-6 stimulated similar IgG secretion from tonsil B cells and also required the presence of IL-2. Moreover, the combination of IL-3 and IL-6 induced IgG secretion equivalent to that induced by either lymphokine. These data suggest that IL-3 and IL-6 might affect normal B cell differentiation by similar mechanism(s). The IL-3 effect on B cells appears to be caused by direct interaction with B cells because IL-3 induced a dose-dependent stimulation of IgG secretion from the JDA cells. This stimulation did not require the presence of IL-2. IL-6 displayed a similar effect on JDA cells and did not require IL-2. However, when IL-3 was combined with IL-6 a synergistic IgG secretion was observed in JDA cultures. These data suggest that IL-3 may potentiate the human immune response via stimulation of B cell differentiation and that its effect is dependent on the target B cell population, its stage of activation and/or maturation.     

Dendritic and B-cell function during antibody responses in normal and immunodeficient (xid) mouse spleen cultures

Dendritic cells (DC) act as accessory cells for T-dependent antibody responses in two ways. One is to induce a class of stimulating factors (BSF) which allow B lymphocytes to respond to heterologous red cells as antigen. xid DC induce the production of these BSF, but xid B cells totally lack responsiveness. A second mechanism of DC function applies to red cell and haptenated-protein antigens. Here DC, helper T lymphocytes, and antigen-specific B cells interact in discrete clusters. Then the B cells become responsive to BSF. xid DC are fully active in this pathway, and xid B cells develop significant (10-20% of control) responses. This partial reduction in xid B-cell function could be due to the poor viability of xid lymphocytes in vitro. There is a comparable reduction in xid polyclonal responses to alloreactive helper T blasts. The other severe deficit in xid involves antibody formation to haptens on polysaccharide carriers. This response in normal mice is not influenced by DC or by BSF. The only similarity between DNP-Ficoll and RBC plus BSF responses is that both utilize B lymphocytes that do not associate with DC-T clusters, even though helper cells for DNP-Ficoll and for RBC are present in the culture. We conclude that DC function is not altered in xid. The main deficit seems to be in a B-cell activation pathway that is shared by polysaccharide carriers and some but not all BSF, and/or in a B-cell subpopulation that does not interact with carrier-specific helper cells. We speculate that this B-cell alteration primarily involves the Ig delta-poor marginal zone subpopulation of splenic B lymphocytes.     

Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor

Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.     

Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop

Mucus hypersecretion and persistent airway inflammation are common features of various airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. One key question is: does the associated airway inflammation in these diseases affect mucus production? If so, what is the underlying mechanism? It appears that increased mucus secretion results from increased mucin gene expression and is also frequently accompanied by an increased number of mucous cells (goblet cell hyperplasia/metaplasia) in the airway epithelium. Many studies on mucin gene expression have been directed toward Th2 cytokines such as interleukin (IL)-4, IL-9, and IL-13 because of their known pathophysiological role in allergic airway diseases such as asthma. However, the effect of these cytokines has not been definitely linked to their direct interaction with airway epithelial cells. In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleukin-1alpha, 1beta, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor alpha). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.     

IL-6 Amplifier, NF-κB-Triggered Positive Feedback for IL-6 Signaling, in Grafts Is Involved in Allogeneic Rejection Responses

The IL-6-amplifier first was discovered as a synergistic activation mechanism for NF-κB/STAT3 in type 1 collagen(+) cells. This process is marked by the hyperinduction of chemokines and subsequent local inflammation that leads to autoimmune diseases. In this study, we show that IL-6 amplifier activation in grafts plays important roles in allogeneic graft rejection by using a tracheal heterotopic transplantation model that includes bronchiolitis obliterans, a pathological marker for chronic rejection. IL-6, epidermal growth factor, and IFN-γ all stimulate IL-6 amplifier activation, whereas CCL2, a chemotactic factor for Th1 cells, was one of the amplifier's main targets. Interestingly, IFN-γ hyperinduced CCL2 in type 1 collagen(+) cells via the IL-6 amplifier at least in vitro. In addition, we detected IL-6, CCL2, phospho-STAT3, and phospho-NF-κB in epithelial type 1 collagen(+) cells of allogeneic tracheal grafts. These results show that IL-6 amplifier activation in grafts plays a critical role for graft rejection responses after allogeneic transplantation, including chronic rejection. From these results, we consider whether the IL-6 amplifier in grafts might be a valuable therapeutic target for the prevention of transplant rejection, including chronic rejection.