Following our previous studies with NKT cell-deficient mice, we demonstrated here that excisional cutaneous wounds exhibit accelerated closure when NKT cell activation is prevented by systemic (i.v.) administration of anti-CD1d mAb ( and ) and that the effects of CD1d were dose-responsive (). This model therefore demonstrates that cutaneous wounding activates NKT cells via
CD1d presentation of glycolipid antigen, and not any of the alternative modes of NKT cell activation (FcR engagement or direct binding of antigen to the invariant TCR). Just as when NKT cells are congenitally absent, preventing NKT cell activation leads to augmented local production of a key subset of neutrophil and monocyte macrophage chemokines (). Despite enhanced wound content of MIP-2, MCP-1, MIP-1α
, and MIP-1β
, inflammatory cell infiltrates in the wounds of IgG and anti-CD1d treated animals remained unchanged ( and ). While somewhat surprising, these observations confirmed our previous observations of alterations in the local inflammatory response and acceleration of wound closure in mice in which NKT cells are functionally impaired or congenitally absent (CD1dko or Jα
]. In other words, our earlier observations in knockout animals were not due to the compensatory response of an immune system lacking a regulatory element.
Increased levels of chemokines without a corresponding increase in inflammatory cell number may seem contradictory. This contradiction can be resolved when one considers that these chemoattractant molecules reach a level of saturation, and a healing wound may already be at or near the threshold for recruitment [39
]. Aside from leukocyte recruitment, the chemokines have a variety of alternative functions. In a healing wound, these include epithelialization, angiogenesis, and fibrosis [39
]. For example, the CXC chemokines such as MIP-2 have been associated with angiogenesis and re-epithelialization [42
], while the CC chemokines, MCP-1 and MIP-1α
, contribute to extracellular matrix deposition and remodeling [45
]. These alternative chemokine functions are consistent with accelerated wound closure, which we also observed when NKT cells are absent [4
] or their activation prevented ( and ).
We also identified the optimal dose and timing of anti-CD1d in a mouse system. A dose of 100 μ
g proved to be most effective as the higher dose, 200 μ
g, resulted in wound areas equivalent to those seen in animals given control IgG (). This higher dose also reduced the numbers of macrophages and B cells likely via
an antibody-dependent cell-mediated cytotoxicity. Macrophages are essential for wound repair, so a reduction in their numbers might counterbalance any benefit gained from preventing NKT cell activation on wound closure [25
]. The schedule to achieve the greatest effect on wound area proved to be a 6-h pretreatment with anti-CD1d (). This probably reflects the very early timing of NKT cell activation and the interval needed for sufficient CD1d blockade at site(s) of NKT cell activation.
Interestingly, pretreatment also dramatically decreased wound NKT cell content (), indicating that ligation of the invariant TCR may influence NKT cell recruitment to sites of cutaneous injury. These findings were supported by the appearance of activated (CD69+) NKT cells both in the circulation and in wounds themselves early (24 h) after injury (). The appearance of activated (CD69+) NKT cells in circulation early after injury was unexpected since we hypothesized that the wound itself was the site of activation. This finding may imply that cutaneous injury revealed self glycolipid systemically or at sites distant to the skin. In addition, we believe that NKT cell activation is not singular in time or place. Cutaneous injury also resulted in elevated expression of CXCR2 on both wound and circulating NKT cells. CXCR2 expression remained high in animals treated with anti-CD1d, but was diminished in animals receiving the control IgG (), suggesting that ligation of invariant TCR may influence CXCR2 expression level or recycling. This is the first demonstration of such a phenomenon in NKT cells. These data suggest that an activation step via
CD1d presentation of glycolipid antigen is required for NKT cells influence at the wound site, and that the events surrounding NKT cell activation are linked to their homing to sites of cutaneous injury. This information could not be gleaned from our earlier work with knockout models [4
Blockade of NKT cell activation also reduced the numbers of NKT cells that infiltrated cutaneous wounds. In a murine model of renal ischemia-reperfusion injury, Li and colleagues similarly found that pretreatment with anti-CD1d likewise dramatically reduced the numbers of NKT cells that infiltrated the re-perfused kidney at 24 h, and protected the renal parenchyma from inflammatory damage [12
]. Just as in our model of cutaneous injury, blockade of NKT cell activation with anti-CD1d reduced the number of infiltrating NKT cells and improved the overall post-injury outcome. It is unclear whether the salutary effects of preventing NKT cell activation in both settings result from simply a diminished local quantity of NKT cells alone, absent NKT cell function locally, or reduced NKT cell function systemically.
Regardless of the exact mechanism that benefits wound closure when NKT cell activation is blocked with anti-CD1d, it appears that NKT cell activation may assist NKT cell recruitment/homing to sites of inflammation. CXCR2 expression on circulating and wound NKT cells also displayed some degree of plasticity in response to blockade of NKT cell activation. Aside from CXCR2, NKT cells also express various receptors for inflammation-related chemokines, including CXCR3, CXCR4, CXCR6, CCR1, CCR2, and CCR5 [27
]. Others have described how NKT cell subsets exhibit differential expression of chemokine receptors [27
]. Furthermore, T cell phenotype (IL-4 versus
IFN-γ producing) also influences chemokine receptor expression patterns. For example, it has been reported that CCR4 is expressed by almost all IL-4 producing CD4+
T cells, while CXCR3 is expressed by IFN-γ producing cells [51
]. While many of these studies involve human lymphocyte subsets, it is intriguing that phenotype determines chemokine receptor expression profile since NKT cell phenotype is determined at the time of activation. Because we were able to modulate NKT cell CXCR2 expression patterns by blocking NKT cell activation, we favor the hypothesis that triggering the invariant TCR modulates the NKT cell’s homing machinery.
The site of NKT cell activation in this excisional punch wound model remains unknown. Qin and colleagues first suggested that only a small subpopulation of NKT cells express CXCR2 [52
]. As Faunce and colleagues have shown, MIP-2 recruited NKT cells to the spleen, where they became activated [26
]. In other words, chemokine recruitment preceded activation. Perhaps, ligation of the invariant TCR favors a net down-regulation of surface CXCR2. Therefore, when NKT cell activation is prevented, the NKT cell continues to recycle its CXCR2 receptors, resulting in greater CXCR2 expression at 1 d post-wounding (). Given the augmented local production of MIP-2 that also occurs with anti-CD1d administration, one might expect the higher ligand quantities to eventually result in less relative expression of the receptor at later time points because of the receptor desensitization and down-regulation [39
]. Our laboratory is currently investigating this possibility as well as the connection between NKT cell activation and chemokine receptor expression.
We also found that CD69 expression by NKT cells in circulation was decreased by pretreatment with anti-CD1d. One potential explanation for these findings is that some subset of NKT cells encounters their endogenous glycolipid antigen prior to entering the wound site. NKT cell activation may not be singular in time or place. Since preventing NKT cell activation via
anti-CD1d diminished expression of CD69 on circulating NKT cells, this suggests that cutaneous injury results in a systemic, rather than a purely local, revelation of glycolipid antigen and subsequent invariant TCR ligation. Although the exact mechanisms behind NKT cell regulation of wound inflammation requires further investigation, the studies presented here demonstrate the powerful therapeutic potential of anti-CD1d in achieving accelerated wound closure. In the clinical setting, achieving faster wound closure greatly benefits patients since it quells the nutritional and metabolic demands of the patient and ends an opportunity for infections. The last 30 y have seen abundant laboratory and clinical testing of the various soluble cytokines and growth factors important in wound repair in an attempt at accelerating wound closure [33
]. Among these, only one (platelet-derived growth factor) has been approved by the Food and Drug Administration (FDA) as a wound healing therapy [57
]. Directing therapy at any single cytokine or growth factor may not overcome evolutionary redundancy and, alternatively, might grossly alter the microenvironment, thereby negating any potential benefit and even possibly driving the process toward pathologic or excessive healing. Instead, targeting a cell type such as the NKT cell rather than a soluble factor might enable a subtler, multi-faceted therapy, since the NKT cell functions by interacting with multiple cell types and secreting a myriad of soluble factors. Hence, a cell-based approach such as targeting NKT cell activation with anti-CD1d might advance an opportunity to more precisely alter the wound microenvironment in a manner that ultimately improves wound healing.