Novobrantseva TI, Majeau GR, Amatucci A, Kogan S, Brenner I, Casola S, Shlomchik MJ, Koteliansky V, Hochman PS, Ibraghimov A. Attenuated liver fibrosis in the absence of B cells. J Clin Invest. 2005 Nov;115(11):3072-82. Reprinted by permission of the American Society for Clinical Investigation.
Analysis of mononuclear cells in the adult mouse liver revealed that B cells represent as much as half of the intrahepatic lymphocyte population. Intrahepatic B cells (IHB cells) are phenotypically similar to splenic B2 cells but express lower levels of CD23 and CD21 and higher levels of CD5. IHB cells proliferate as well as splenic B cells in response to anti-IgM and LPS stimulation in vitro. VDJ gene rearrangements in IHB cells contain insertions of N,P region nucleotides characteristic of B cells maturing in the adult bone marrow rather than in the fetal liver. To evaluate whether B cells can have an impact on liver pathology, we compared CCl4-induced fibrosis development in B cell-deficient and wild-type mice. CCl4 caused similar acute liver injury in mutant and wild-type mice. However, following 6 weeks of CCl4 treatment, histochemical analyses showed markedly reduced collagen deposition in B cell-deficient as compared with wild-type mice. By analyzing mice that have normal numbers of B cells but lack either T cells or immunoglobulin in the serum, we established that B cells have an impact on fibrosis in an antibody- and T cell-independent manner.
The fibrosis of chronic liver disease is characterized by iterative cycles of tissue injury, inflammation and repair associated with a chronic persistent inflammatory infiltrate. Although previously considered irreversible, clinical observations and studies in animal models have demonstrated that even advanced fibrosis can resolve under some circumstances.1 Liver fibrosis is mediated by activated hepatic stellate cells (HSC) which adopt a myofibroblast phenotype in response to injury and regulate matrix remodelling and fibrosis. Changes in the tissue microenvironment promote chronic inflammation by skewing the local homeostatic balance toward inflammatory cell recruitment, retention and survival rather than apoptosis and resolution.2
Regression of fibrosis in animal models is prefaced by apoptosis of HSC followed by resolution of the inflammatory infiltrate,1 demonstrating that fibrosis is not a simple hierarchical process but the result of complex paracrine and autocrine interactions between stromal cells, ECM and leukocytes. Although fibroblasts and macrophages are central to the pathogenesis of liver fibrosis, increasing attention has been paid to the role of other leukocytes, and the recent paper by Novobratseva et al.in JCI suggests that B cells can be added to the list of pro-fibrogenic effector cells. The complex role played by macrophages is highlighted by studies demonstrating that subsets of these cells within tissue play distinct roles in promoting HSC dependent scarring and regression of fibrosis.3 Macrophages can maintain profibrotic responses and TH2 polarisation by secreting cytokines such as IL-6 and TGFβ, which promote HSC proliferation and fibrogenesis.4 Regulatory B cells have also been shown to direct the polarity of the local inflammatory response away from a TH1-like phenotype in autoimmunity and may contribute to local production of TGFβ1.5, 6 The contribution of intra-hepatic T cells to fibrogenesis has been reported in several studies. Mice rendered lymphopenic by sub-lethal irradiation and RAG2−/− knockout mice, which lack T and B cells, are both resistant to CCL4 induced fibrosis7, 8 and adoptive transfer studies in a variety of murine models have suggested a role for CD4+ and CD8+ T cells. CD4+ T cells were originally identified as the source of a secreted “fibrotic factor” in a mouse model of liver fibrosis which was subsequently shown to be IL-13.9
The polarity of the inflammatory response is critical for fibrogenesis, and different programs of gene expression are induced when chronic inflammatory responses are dominated by TH1 or TH2 cytokines. Although TH1 cytokines characteristically generate a rapid and intense inflammatory response they cause little fibrosis,10 whereas TH2 cytokines increase transcription of several genes involved in the fibrotic process, e.g., pro-collagen I and III, MMP2, MMP9 and TIMPs,4 all of which are expressed in CCl4 induced hepatitis, suggesting that mechanisms of fibrosis may share a common cytokine profile. Further evidence of the importance of TH2 cytokines in fibrogenesis is demonstrated by studies using mice that lack the TH2 cytokine IL-4 or its receptor IL-4Ra; or those with a targeted disruption of STAT6, the transcription factor which drives many TH2 responses. These animals show reduced fibrosis in a murine model of scleroderma11 although interestingly in light of the paper by Novobratseva, B cell deficient mice showed normal fibrotic responses in this model.12 Safadi et al. demonstrated a role for CD8+ but not CD4 T-cells in hepatic fibrogenesis in response to CCl4 or thioacetamide in SCID mice.7 There is therefore good evidence4 that T cells are important contributors to the fibrotic process, although their precise role remains unclear.
The role of B-lymphocytes in these processes is poorly understood, but their ability to activate T-cells at low antigen loads and to secrete a wide range of cytokines suggests they could also be involved in the perpetuation of inflammatory responses in fibrotic tissue. The paper by Novobrantseva et al. provides the first direct evidence of a role for B cells in liver fibrosis. Using BALB/c and C57BL/6 mice they phenotyped the resident lymphocyte population of murine livers and then examined the fibrotic response to CCL4 in B and T lymphocyte deficient mice. They found that around 50% of the intra-hepatic lymphocytes were CD19+ B lymphocytes and detailed phenotyping and functional studies revealed that they were similar to activated splenic B2 cells. Genetic analysis of their VDJ gene rearrangements showed that intrahepatic B cells contain insertions of N,P region nucleotides characteristic of B cells that have matured in the bone marrow rather than the foetal liver. These observations suggest that murine intrahepatic B-cells do not originate in embryonic liver, but are the product of selective recruitment from the peripheral B-cell pool13 with possible further activation within the liver.
How do these specialized B-cells interact with other leukocytes following liver injury and can they modulate fibrosis in a manner previously thought to be the preserve of T cells? To answer these questions Novobratseva et.al. studied CCL4induced fibrosis in B cell (JH− /−) and T cell deficient mice. Although the acute inflammatory response to repeated injections of CCL4 was the same in wild type and JH− /− mice, fibrosis was markedly reduced in the B cell deficient animals after 6 injections of CCl4. This was not the result of an impaired inflammatory response because both B cell deficient animals and controls showed similar necro-inflammatory damage 24 hours after CCl4injection. They found similar results in mice treated with ANIT, a hepatotoxic agent that induces neutrophil-dependent liver necrosis, demonstrating that B cell deplete mice show reduced fibrotic responses following repeated liver injury in at least two experimental models. These results illustrate the maxim that it isn't how much but rather which type of inflammation that determines whether injury progresses to fibrosis.
JH−/− mice display extensive immune defects; they lack follicular dendritic networks in lymph node follicles and follicular associated epithelium in gut associated lymphoid tissue, both of which are important sites of B cell/T cell interaction; they also show impaired CD4 and NKT function. To investigate the role of other lymphocyte subsets in the fibrotic response of these animals, the authors repeated the experiments using combinations of CD4, CD8 and γδ T cell deficient mice as well as RAG2− /− mice. Only the RAG2− /− mouse, an animal lacking both T and B cells, showed a reduction in fibrosis and T cell deficient animals were not protected, emphasising the importance of B cell mediated immune responses in this model.
B cells could directly modify fibrotic responses in a T cell independent manner by secreting immunoglobulins or cytokines, or through contact-dependent cross-talk with fibroblasts, macrophages and NK subsets. The authors show that B-cell conditioned media promotes collagen synthesis in cultured fibroblasts but when experiments were repeated using mice unable to secrete immunoglobulin no effect was seen. Thus, a secreted factor is involved but the underlying mechanism is antibody independent. The fact that T cell deficient mice showed normal fibrotic responses in this model argues against a role for B cells in antigen presentation. Therefore, it is most likely that B cells promote fibrosis by secreting cytokines or by contact-dependent interactions with other cells that favour a pro-fibrotic microenvironment. Differentiation of naïve B cells into TH1-like or TH2-like patterns of cytokine secretion is determined by similar cytokine microenvironments to those that regulate corresponding T cell differentiation, and B-cells secrete the pro-fibrotic cytokines IL-4, IL-6, and IL-13 at levels similar to CD4+ T-cells.14 Whilst BCR ligation is required for optimal cytokine secretion, repetitive BCR-independent stimulation is sufficient to stimulate naive B cells to secrete cytokines in a polarised fashion.15, 16 Repeated cycles of tissue injury may therefore provide stimulatory signals to maintain B-cell activation, thereby allowing the process to become self-perpetuating. Interactions between NKT cells, macrophages and B-cells within this fibrotic environment will also provide directional signals that reinforce local cellular responses and secretion of TGFβ,17 emphasising the importance of local cell networks in providing the context for cytokine expression in fibrotic tissue (Fig. 1).
Co-stimulatory signals provided through direct cell-cell contact with T-cells, NKTs, Kupffer cells and mast cells will also be important. CD40 is a strong candidate for a central role in these paracrine interactions. CD40L is strongly expressed on lymphocytes and macrophages/Kupffer cells at sites of liver injury in several diseases.18 This cellular source of CD40L could activate B cell CD40 resulting in B cell survival, growth, differentiation and IL-6 secretion, thereby providing local paracrine signals to induce and sustain HSC proliferation and collagen synthesis. To avoid dysregulated bystander activation of B cells, upregulation of CD40 in B cells is usually accompanied by increased surface expression of Fas that limits B cell proliferation.19 It is very likely that survival signals (such as BAFF/BLyS) displayed in the fibrotic microenvironment may overcome this regulatory mechanism, allowing inappropriate retention and survival of effector B-cells, thus perpetuating inflammation and amplifying the local fibrotic response.2
So what messages can we take from this work? Clearly B cells need to be considered as another cell type capable of modulating fibrosis in response to tissue injury. This suggests that B cells or B cell derived cytokines could be attractive new targets for anti-fibrotic therapy and the increasing availability of biologic agents which target B cells or B cell cytokines should allow this approach to be tested in humans. Chronic HCV infection is associated with the presence of intrahepatic follicles that contain activated B cells, and some patients develop B cell lymphomas expressing HCV-specific antigen receptors, suggesting a role for chronic antigenic stimulation in their development. Such observations suggest that HCV proteins can directly modulate B cell function. If the conclusions of the present study are correct, then B cell activation in HCV might have direct profibrotic effects as well as operating through antibody or immune complex dependent mechanisms.
Although these data are thought provoking, one must be cautious about translating conclusions reached in animal models to humans. The lymphocyte composition of mouse and human livers differ with different proportions of CD4/CD8 T cells, NK, NKT and γδ T-cells.20 Furthermore B cells are found in much smaller proportions in humans, typically about 5% to 10% of the total liver lymphocyte pool, and possess less distinct B cell subsets. The relevance of these differences for fibrogenesis is not known but they stress the need for confirmation of the animal data by studying human disease or using human in vitro models.