These studies advanced insights into innate responses following cell transplantation in the liver. First, profiling of chemokines and cytokines offered clues to the nature of inflammatory cells recruited by cell transplantation, i.e., neutrophils and Kupffer cells. Second, depletion of neutrophils and Kupffer cells permitted dissection of inflammatory mechanisms induced by cell transplantation. Third, suppression of chemokine and cytokine expression after depletion of neutrophils and Kupffer cells improved engraftment of hepatocytes in the liver.
Here, we limited studies of allogeneic cell transplants to the short-term and avoided xenogeneic cell transplants, which additionally activate a variety of adaptive immune responses.12,13
By contrast, transplantation of syngeneic cells permitted analysis of the innate immune system, which proved important in the initial clearance and engraftment of transplanted cells. Transplantation of autologous hepatocytes will be appropriate in some conditions for cell therapy with genetically-modified hepatocytes, as was examined in familial hypercholesterolemia.14
Also, suitable autologous cells, e.g., those derived from person-specific induced stem cells, will eventually be of interest for cell therapy. Of course, allogeneic and xenogeneic donor cells are of considerable interest for cell therapy, although analysis of relevant cytokine-chemokine-dependent pathways in such cell transplants will require detailed further work, where our studies here should provide an excellent comparison. Our targeted array-based approach to elucidate cytokine-chemokine changes was effective. High-density microarrays to study genome-wide transcriptional changes offer an alternative approach,15
although this approach generates far larger datasets, requiring more complex analyses, and our conclusions regarding inflammatory mechanisms would likely not have changed.
Neutrophils and Kupffer cells emerged in our studies as predominant effectors of cell transplantation-induced early inflammation. Increased expression of CCL3, CCL4, CXCL1 and CXCL2, which are released from macrophages, neutrophils or epithelial cells, 16,17
was in agreement with recruitment of neutrophils and Kupffer cells. CCL3 binds CCR1 and CCR5, CCL4 binds CCR5, and CXCL1 and CXCL2 bind CXCR2, as well as other receptors ().6
The potency of CXCL1 and CXCL2 in attracting neutrophils to sites of inflammation is well established,18
whereas CCL3 is chemotactic for monocytes, eosinophils and basophils.19
CXCL4 is expressed in activated platelets with additional roles in attracting neutrophils, monocytes or lymphocytes, although its receptor, CXCR3, was not overexpressed after syngeneic cell transplantation.20,21
Our results suggested potential roles for additional chemokines. For instance, CCL17, CCL22, CXCL9 or CXCL10 are released by macrophages, as well as eosinophils.22
CXCL10 may additionally be released from endothelial cells,23
which should be relevant since LSEC are damaged by cell transplantation.2
Similarly, Bcl6 (B-cell leukemia/lymphoma 6 protein) and its receptor CXCR5, were expressed after cell transplantation. Bcl6 is required for the function of germinal B cells though not for differentiation of memory B cells.15,24
Our histological studies did not demonstrate accumulation of eosinophils or basophils and we did not identify accumulation of lymphocytes or NK cells in the liver, which was in agreement with their playing little or no roles in this setting of hepatic inflammation after syngeneic cell transplants.
Our studies with anti-PMN and GdCl3 substantiated the role of neutrophils and Kupffer cells in the inflammatory response after cell transplantation. Simultaneous depletion of neutrophils and Kupffer cells virtually abolished the early inflammatory response after cell transplantation. Crosstalk between neutrophils and Kupffer cells likely accounted for these changes in chemokine expression. Depletion of neutrophils with anti-PMN before cell transplantation decreased CXCL1 expresssion, although CXCL2 expression increased, suggesting that the latter was additionally expressed elsewhere. In GdCl3-treated animals, expression of CXCL1, as well as CXCL2 increased by 60- and 457-fold, respectively, likely due to expression from neutrophils. By contrast, neutrophil depletion had smaller effects on CCL3 and CCL4 expression compared with normalization of their expression after Kupffer cell depletion, consistent with their production in the latter. Expression of CCR1, CCR2, CXCR1, CXCR2 and CXCR5 was normal under both conditions.
This activation of neutrophils and Kupffer cells after cell transplantation was in agreement with their roles in other forms of liver injury, e.g., ischemia-reperfusion injury, an inevitable additional accompaniment of hepatocyte transplantation.25
This type of liver injury occurs in phases, where Kupffer cells mediate the initial response through proinflammatory cytokines, e.g., TNF-α or IL-6, in association with neutrophils.26
Our data showing greater expression of TNF-α and its receptor, TNFrSF1b, as well as the associated genes Ltb and ABCF1, which participate in TNF-α-induced inflammation,27,28
along with normalization of this TNF-α group only after anti-PMN, indicated that these changes were due to neutrophil activation. Moreover, neutrophils may activate release of TNF-α and IL6 from macrophages.29
Also, TNF-α may induce expression of multiple chemokines by itself from parenchymal cells, e.g., CCL3, CCL4, as well as CCR1 and CCR2, 30
or in cooperation with other cytokines, e.g., TNF-α induced expression of CCL9, CCL10, CCL17, and CCL22 in the presence of IL4 or interferon-γ. 22,23
Therefore, TNF-α is likely to have played a major role in liver inflammation after cell transplantation. Similarly, upregulation of an IL6 receptor along with IL6 signal transducer, and of IL1 receptor along with tollip, 31
should indicate roles for these cytokines. Increased availability of C3 complement component and integrin beta2 was probably helpful in directing chemokines to suitable sites.6,32
Proteolytic cleavages of chemokines by matrix metalloproteinases, which are expressed after cell transplantation,4
may promote their chemokine activity.6
These mechanisms of inflammation should be highly significant for defining organ-specific perturbations after cell transplantation and for guiding strategies in cell and gene therapy. The role of chemokine receptors, such as CCR1, CCR2, CCR5 and CXCR3, in allografts has been studied in knockout mice for tissue (pancreatic islets), epithelium (cornea) or solid organ (cardiac) transplantation.33–35
The precise mechanisms by which the inflammatory environment of the liver following cell transplantation may impair cell engraftment are unknown at present. However, cell isolation with collagenase digestion produces significant perturbations in hepatocytes. While exposure of such hepatocytes to discrete pro-inflammatory cytokines, e.g., TNF-α, by itself, is insufficient for cell necrosis or apoptosis, 36
the combination of multiple cytokine-chemokines in the presence of additional perturbations related to ischemia or other hepatic changes may cause cytotoxicity. Our studies offer several chemokine and cytokine targets that can be directly blocked with drugs to demonstrate changes in cell engraftment and for improving outcomes in cell therapy. Among potential drug targets should include receptors, such as CXCR2 to block neutrophil-specific activities, and CCR1, CCR2 or CCR5 to block Kupffer cell or monocyte-specific activities. Similarly, blockade of TNF-α should offer suitable means to overcome additional inflammatory perturbations. Targeting of specific molecules should produce lesser general toxicity, as observed with the combination of anti-PMN plus GdCl3
in our studies. As chemokines and cytokines are naturally involved in allograft responses through NK, dendritic, antigen-presenting, and T and B cells, transplantation of allogeneic or xenogeneic cells will likely benefit from these insights, although further work will be necessary to establish the significance of additional cytokine-chemokine changes following such cell transplants.