No abstract

Abstract. Interleukin (IL)-8 is a pro-inflammatory cytokine that has a direct effect on immune cells, including polymorphonuclear cells. Keratinocytes are a rich source of IL-8. However, there is little knowledge on the role of IL-8 in clinical wound healing and the direct biological effect of IL-8 on keratinocytes. In this study, the effect of recombinant human IL-8 (rhIL-8) on migration and adhesion was tested using HaCaT keratinocytes as a cell model. The cell functions were evaluated using impedance cell sensing. The expression of IL-8 receptor (IL-8R) transcripts in human skin and wounds (acute and chronic) was assessed using real-time transcript analysis. rhIL-8 significantly increased the migration of keratinocytes (3.5±0.3 for cells treated with IL-8 vs. 2.7±0.6 for controls; p= 0.029). It is interesting to note that treatment of keratinocytes with IL-8 resulted in a marked shift in the responsive frequencies. IL-8 only resulted in a marginal increase in cell adhesion, which was particularly noticeable at high frequencies. The PLC-γ inhibitor completely eradicated the action of IL-8 on the migration of HaCaT cells. Using real time PCR, it was found that chronic wounds had significantly lower levels of the B form of the IL-8R (IL-8RB) (p=0.045) and marginally lower levels of the A form, IL-8RA, in comparison with acute wounds. Therefore, IL-8 has a direct and profound stimulatory effect on the migration of human keratinocytes, which is likely to occur via the PLC-γ pathway. Together with a reduced level of IL-8Rs in difficult-healing wounds, IL-8 has a clear prognostic and therapeutic value in wound healing.

These data indicate that human wound-associated lymphocyte populations are modulated during healing; the increase in numbers of CD8+ T-suppressor lymphocytes is in accordance with previous animal data, indicating a role for these cells in downregulating healing as the wound closes. This study also documents an associated increase in B lymphocytes and healing of human wounds, with an as yet undefined role.

Oligodendrocytes were studied in the anterior medullary velum (AMV) of the rat using the monoclonal antibody Rip, an oligodendrocyte marker of unknown function. Confocal microscopic imaging of double immunofluorescent labelling with antibodies to Rip and carbonic anhydrase II (CAII) revealed two biochemically and morphologically distinct populations of oligodendrocyte which were either Rip+CAII+ or Rip+CAII-. Double immunofluorescent labelling with Rip and myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) provided direct evidence that Rip-labelled cells were phenotypically oligodendrocytes and confirmed that Rip did not recognise astrocytes. Oligodendrocytes which were Rip+CAII+ supported numerous myelin sheaths for small diameter axons, whilst Rip+CAII- oligodendrocytes supported fewer myelin sheaths for large diameter axons. Morphologically, Rip+CAII+ oligodendrocytes corresponded to types I or II of classical nomenclature, whilst Rip+CAII- oligodendrocytes corresponded to types III and IV. The results demonstrated a biochemical difference between oligodendrocytes which myelinated small and large diameter fibres.

The objective of this study was to characterize the leucocyte infiltrate which accumulates at the margin of chronic wounds. These leucocytes are a rich source of cytokines and growth factors, and an inappropriate function of these cells may contribute to the maintenance of wound chronicity. The leucocyte populations were stained immunohistochemically with monoclonal antibodies specific for surface receptors which give an indication of cellular function. Wound margin biopsies taken from chronic leg ulcers exhibited a localized infiltrate of CD45+ leucocytes associated with vascularized tissue in the dermis adjacent to the wound margin. Lymphocytes were identified in highest numbers in this area and CD45RO+ T lymphocytes predominated over B lymphocytes, which were either absent or present in very low numbers. In the majority of chronic wounds examined, CD4+ T lymphocytes were present in greater numbers than CD8+ T lymphocytes with a mean (+/-SD) ratio of CD4+:CD8+ of 1.5 +/- 0.6. CD68+ macrophages were identified in all layers of the dermis at the chronic wound margin. In 60% of wounds examined, macrophages were negative for the activation associated markers CD16 (Fc gamma III receptor) and CD35 (C3b receptor). In those biopsies where CD16 and CD35 positive macrophages were observed these were preferentially located in the perivascular regions. These data indicate that as monocytes extravasate into chronic wound tissue they may be subjected to microenvironmental influences which either suppress or do not induce macrophage activation. Suppression of macrophage activation may lead to an inappropriate cytokine/growth factor secretion and contribute to the maintenance of wound chronicity.

The anterior medullary velum is a thin sheet of CNS tissue which roofs the rostral part of the IVth ventricle and contains fascicles of myelinated fibres which, in part, arise from the nucleus of the IVth cranial nerve. This study used histochemical, immunohistochemical, and intracellular dye-injection techniques to describe cellular interrelationships in the velum in whole-mounts and in sections. Rip antibody-stained whole mounts provided a unique description of both oligodendrocyte units (defined as an oligodendrocyte and the complement of myelinated internodal segments it forms), and consecutive myelin sheaths along the same axon. A broad range of unit morphologies was categorised into four arbitrary groups, according to classical criteria, which comprised small cells supporting the short, thin myelin sheaths of 15-30 small diameter axons (Type I), through intermediate types (II & III), to the largest cells forming the long, thick myelin sheaths of 1-3 large diameter axons. Rip antibody and ferric ion-ferrocyanide staining, together with intracellular dye injection, revealed oligodendrocyte process branching patterns and their mode of engagement of myelin sheaths, nodes of Ranvier, and the spatial disposition of the outer cytoplasmic rims of myelin sheaths. The latter formed a conspicuous spiral ridge on the exterior surface of myelin sheaths which connected with the paranodal loops at each heminode. Large bundles of axons decussated through the velum, the bulk of which were IVth nerve fibres which constituted the IVth nerve rootlet. The PNS/CNS transitional zone of the IVth nerve was located 0.25-0.50 mm along the root, where astrocytic end-feet defined an abrupt margin, convex towards the periphery, where the heminodes of central and peripheral myelin were apposed, and where the basal lamina tubes of the Schwann cell units were discontinued. The basal processes of ependymal cells lining the ventricular wall of the velum, passed between axon bundles before abutting on the basal lamina of the pia. Many of these processes branched and ran along the axonal bundles. A monolayer of microglia occupied a subependymal stratum in which the non-overlapping dendritic territories of each cell formed a regular mosaic throughout the velum without any obvious interaction with either axons or other glial cells. Astrocytes were also uniformly distributed; their fine processes made up a dense lattice amongst axons, often running parallel and within the fibre bundles; stouter ones had terminal end-feet which undercoated the basal lamina of both the glia limitans externa and the blood vessels in the velum.

Defolliculated (Gsdma3(Dfl)/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3(Dfl)/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1-/- mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies.

Interleukin (IL)-24, also known as melanoma differentiation-associated gene-7, is a cytokine initially identified from cancerous cells and expressed in a range of cell types. It is a regulator of cell differentiation, growth, and apoptosis, and a promising anticancer agent. IL-24 acts via its heterodimic receptors: the IL-20R1 and IL-20R2 complex and the IL-22R1 and IL-20R2 complex. There is limited information on the effect of IL-24 in wound healing. Human acute and chronic wound tissues were used to analyze the transcript levels and histological staining of IL-24 and the IL-24 receptors. The biological response of human keratinocytes to recombinant human IL-24 was evaluated using electric cell-substrate impedance sensing-based methods in conjunction with inhibitors to candidate signaling pathways. IL-24 significantly slowed the migration of keratinocytes (p = 0.01), with only a marginal effect on cellular adhesion. The inhibitory effect of IL-24 on migration was completed reversed following addition of an AKT inhibitor (p = 0.004) but not an SMAD3 pathway inhibitor. Human chronic wound tissues showed raised levels of both IL-24 (p = 0.003) and its receptor (p = 0.0305) compared with acute wound tissues. We conclude that IL-24 appears to promote wound chronicity via its inhibitory effect on the migratory behavior of human keratinocytes, mediated through an AKT-dependent pathway.