The Effect of Radiation Treatment of Solid Tumors on Neutrophil Infiltration and Function: A Systematic Review.

Radiation therapy (RT) initiates a local and systemic immune response which can induce antitumor immunity and improve immunotherapy efficacy. Neutrophils are among the first immune cells that infiltrate tumors after RT and are suggested to be essential for the initial antitumor immune response. However, neutrophils in tumors are associated with poor outcomes and RT-induced neutrophil infiltration could also change the composition of the tumor microenvironment (TME) in favor of tumor progression. To improve RT efficacy for patients with cancer it is important to understand the interplay between RT and neutrophils. Here, we review the literature on how RT affects the infiltration and function of neutrophils in the TME of solid tumors, using both patients studies and preclinical murine in vivo models. In general, it was found that neutrophil levels increase and reach maximal levels in the first days after RT and can remain elevated up to 3 weeks. Most studies report an immunosuppressive role of neutrophils in the TME after RT, caused by upregulated expression of neutrophil indoleamine 2,3-dioxygenase 1 and arginase 1, as well as neutrophil extracellular trap formation. RT was also associated with increased reactive oxygen species production by neutrophils, which can both improve and inhibit antitumor immunity. In addition, multiple murine models showed improved RT efficacy when depleting neutrophils, suggesting that neutrophils have a protumor phenotype after RT. We conclude that the role of neutrophils should not be overlooked when developing RT strategies and requires further investigation in specific tumor types. In addition, neutrophils can possibly be exploited to enhance RT efficacy by combining RT with neutrophil-targeting therapies.

Chronic radiation exposure aggravates atherosclerosis by stimulating neutrophil infiltration.

BACKGROUND: Radiation exposure is known to increase the risk of chronic inflammatory diseases, such as atherosclerosis, by modulating inflammation. METHODS: To investigate the infiltration of leukocytes in radiation-aggravated atherosclerosis, we examined low-density lipoprotein receptor-deficient (Ldlr-/-) mice and C57BL/6j mice after exposure to 0.5 or 1 Gy radiation over 16 weeks. RESULTS: We found that radiation exposure induced atherosclerosis development in Ldlr-/- mice, as demonstrated by increased lipid-laden plaque size, reactive oxygen species levels, and levels of the pro-inflammatory cytokines, IL-1ß and TNF-α, in the aortas and spleens. Total plasma cholesterol, triglyceride, and LDL cholesterol levels were also increased by radiation exposure, along with cardiovascular risk. We also showed dose-dependent increases in neutrophils and monocytes that coincided with a reduction in lymphocytes in the spleens of Ldlr-/- mice. The correlation between the infiltration of leukocytes and cytokine production was also confirmed in the hearts and spleens of these mice. CONCLUSIONS: We concluded that chronic radiation exposure increased the production of pro-inflammatory mediators, which was associated with the migration of neutrophils and inflammatory monocytes into sites of atherosclerosis. Thus, our data suggest that the accumulation of neutrophils and inflammatory monocytes, together with the reduction of lymphocytes, contribute to aggravated atherosclerosis in Ldlr-/- mice under prolonged exposure to radiation.

Neutrophil infiltration and whole-cell vaccine elicited by N-dihydrogalactochitosan combined with NIR phototherapy to enhance antitumor immune response and T cell immune memory.

Melanoma is one of the deadliest malignancies with a high risk of relapse and metastasis. Long-term, tumor-specific, and systemic immunity induced by local intervention is ideal for personalized cancer therapy. Laser immunotherapy (LIT), a combination of local irradiation of laser and local administration of an immunostimulant, was developed to achieve such an immunity. Although LIT showed promising efficacy on tumors, its immunological mechanism is still not understood, especially its spatio-temporal dynamics. Methods: In this study, we investigated LIT-induced immunological responses using a 980-nm laser and a novel immunostimulant, N-dihydrogalactochitosan (GC). Then we followed the functions of key immune cells spatially and temporally using intravital imaging and immunological assays. Results: Immediately after LIT, GC induced a rapid infiltration of neutrophils which ingested most GC in tumors. The cytokines released to the serum peaked at 12 h after LIT. Laser irradiations produced photothermal effects to ablate the tumor, release damage-associated molecular patterns, and generate whole-cell tumor vaccines. LIT-treated tumor-bearing mice efficiently resisted the rechallenged tumor and prevented lung metastasis. Intravital imaging of tumor at rechallenging sites in LIT-treated mice revealed that the infiltration of tumor-infiltrating lymphocytes (TILs) increased with highly active motility. Half of TILs with arrest and confined movements indicated that they had long-time interactions with tumor cells. Furthermore, LIT has synergistic effect with checkpoint blockade to improve antitumor efficacy. Conclusion: Our research revealed the important role of LIT-induced neutrophil infiltration on the in situ whole-cell vaccine-elicited antitumor immune response and long-term T cell immune memory.

Protective Effect of Metformin, Resveratrol and Alpha-lipoic Acid on Radiation- Induced Pneumonitis and Fibrosis: A Histopathological Study.

BACKGROUND: Radiation-induced pneumonitis and fibrosis are the most common side effects of chest radiotherapy. They result from massive and chronic production of Reactive Oxygen Species (ROS), inhibition of antioxidant enzymes as well as the release of several inflammatory mediators. In this study, we aimed to detect the radioprotective effects of metformin (as inhibitor of mitochondrial ROS), resveratrol (as stimulator of antioxidant defense enzymes) and alpha-lipoic acid (as direct antioxidant) for alleviating radiation-induced pneumonitis and fibrosis. METHODS: 80 Male Mice were randomly allotted to eight groups which include G1: control; G2: resveratrol; G3: alpha-lipoic acid; G4: metformin; G5: radiation; G6: radiation plus resveratrol; G7: radiation plus alpha-lipoic acid; G8: radiation plus metformin. Drugs' doses were as follows: 100 mg/kg metformin, 200 mg/kg resveratrol and 200 mg/kg alpha-lipoic acid. Irradiation with a single radiation dose of 18 Gy was performed using a cobalt-60 (60Co) gamma-ray source. After 80 days, all mice were sacrificed and their lung tissues evaluated for morphological changes using histopathological markers. RESULTS: Irradiation led to acute pneumonitis including infiltration of inflammatory cells and damages to alveolar and vascular, as well as mild fibrosis. Metformin, alpha-lipoic acid and resveratrol were able to reduce pneumonitis and overcome radiation-induced fibrosis. CONCLUSION: All agents could protect against radiation-induced lung injury moderately. It is possible that administering higher doses of these drugs over a long period of time could give better radioprotection of the lung.

Alterations in pectoralis muscle cell characteristics after radiation of the human breast in situ.

The life-time risk of being diagnosed with breast cancer is ~12%, hence breast cancer is by far the most common cancer among women. The multimodal treatment concept of breast cancer often intends radiation. The utilized ionizing radiation leads changes in the tissue resulting in tissue damage due to an alteration of molecular factors. The goal of this study was to identify the role of muscle-catabolic proteins after radiation of human pectoralis major muscles in situ. Tissue of the pectoralis major muscle was collected in 12 breast cancer patients after radiation (maximum 3 years after radiation) undergoing a deep inferior epigastric perforator free-flap breast reconstruction. At the same time, an intraindividual comparison to rectus abdominis muscle was carried out upon free-flap elevation. Immunological properties, cell proliferation, differentiation as well as the expression profile of the muscle tissue were investigated through immunohistological reactions, a DNA-microarray and histology. We found significantly increased neutrophil immigration in the radiated muscle tissue. At the same time, proteins responsible for muscular atrophy and apoptosis were significantly elevated in immunohistochemistry. A DNA microarray detected immunological upregulation and myo-differentiative disorders in radiated muscle tissue. This novel study investigating catabolism in radiated muscle in situ can serve as a basis for the treatment of radiation-accompanied muscle disorders.

Total Body Irradiation Mitigates Inflammation and Extends the Therapeutic Time Window for Anti-Ricin Antibody Treatment against Pulmonary Ricinosis in Mice.

Ricin, a highly toxic plant-derived toxin, is considered a potential weapon in biowarfare and bioterrorism due to its pronounced toxicity, high availability, and ease of preparation. Pulmonary exposure to ricin results in the generation of an acute edematous inflammation followed by respiratory insufficiency and death. Massive neutrophil recruitment to the lungs may contribute significantly to ricin-mediated morbidity. In this study, total body irradiation (TBI) served as a non-pharmacological tool to decrease the potential neutrophil-induced lung injury. TBI significantly postponed the time to death of intranasally ricin-intoxicated mice, given that leukopenia remained stable following intoxication. This increase in time to death coincided with a significant reduction in pro-inflammatory marker levels, and led to marked extension of the therapeutic time window for anti-ricin antibody treatment.

Enhanced sensitivity of Neil1-/- mice to chronic UVB exposure.

Oxidative stress and reactive oxygen species (ROS)-induced DNA base damage are thought to be central mediators of UV-induced carcinogenesis and skin aging. However, increased steady-state levels of ROS-induced DNA base damage have not been reported after chronic UV exposure. Accumulation of ROS-induced DNA base damage is governed by rates of lesion formation and repair. Repair is generally performed by Base Excision Repair (BER), which is initiated by DNA glycosylases, such as 8-oxoguanine glycosylase and Nei-Endonuclease VIII-Like 1 (NEIL1). In the current study, UV light (UVB) was used to elicit protracted low-level ROS challenge in wild-type (WT) and Neil1-/- mouse skin. Relative to WT controls, Neil1-/- mice showed an increased sensitivity to tissue destruction from the chronic UVB exposure, and corresponding enhanced chronic inflammatory responses as measured by cytokine message levels and profiling, as well as neutrophil infiltration. Additionally, levels of several ROS-induced DNA lesions were measured including 4,6-diamino-5-formamidopyrimidine (FapyGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyAde), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxyuracil (5,6-diOH-Ura) and thymine glycol (ThyGly). In WT mice, chronic UVB exposure led to increased steady-state levels of FapyGua, FapyAde, and ThyGly with no significant increases in 8-OH-Gua or 5,6-diOH-Ura. Interestingly, the lesions that accumulated were all substrates of NEIL1. Collectively, these data suggest that NEIL1-initiated repair of a subset of ROS-induced DNA base lesions may be insufficient to prevent the initiation of inflammatory pathways during chronic UV exposure in mouse skin.

Synchrotron microbeam irradiation induces neutrophil infiltration, thrombocyte attachment and selective vascular damage in vivo.

Our goal was the visualizing the vascular damage and acute inflammatory response to micro- and minibeam irradiation in vivo. Microbeam (MRT) and minibeam radiation therapies (MBRT) are tumor treatment approaches of potential clinical relevance, both consisting of parallel X-ray beams and allowing the delivery of thousands of Grays within tumors. We compared the effects of microbeams (25-100 µm wide) and minibeams (200-800 µm wide) on vasculature, inflammation and surrounding tissue changes during zebrafish caudal fin regeneration in vivo. Microbeam irradiation triggered an acute inflammatory response restricted to the regenerating tissue. Six hours post irradiation (6 hpi), it was infiltrated by neutrophils and fli1a(+) thrombocytes adhered to the cell wall locally in the beam path. The mature tissue was not affected by microbeam irradiation. In contrast, minibeam irradiation efficiently damaged the immature tissue at 6 hpi and damaged both the mature and immature tissue at 48 hpi. We demonstrate that vascular damage, inflammatory processes and cellular toxicity depend on the beam width and the stage of tissue maturation. Minibeam irradiation did not differentiate between mature and immature tissue. In contrast, all irradiation-induced effects of the microbeams were restricted to the rapidly growing immature tissue, indicating that microbeam irradiation could be a promising tumor treatment tool.

Recombinant Thrombomodulin (Solulin) Ameliorates Early Intestinal Radiation Toxicity in a Preclinical Rat Model.

Intestinal radiation toxicity occurs during and after abdominopelvic radiotherapy. Endothelial cells play a significant role in modulating radiation-induced intestinal damage. We demonstrated that the endothelial cell surface receptor thrombomodulin (TM), a protein with anticoagulant, anti-inflammatory and antioxidant properties, mitigates radiation-induced lethality in mice. The goal of this study was to determine whether recombinant TM (Solulin) can protect the intestine from toxicity in a clinically relevant rat model. A 4 cm loop of rat small bowel was exposed to fractionated 5 Gy X radiation for 9 consecutive days. The animals were randomly assigned to receive daily subcutaneous injections of vehicle or Solulin (3 mg/kg/day or 10 mg/kg/day) for 27 days starting 4 days before irradiation. Early intestinal injury was assessed two weeks after irradiation by quantitative histology, morphometry, immunohistochemistry and luminol bioluminescence imaging. Solulin treatment significantly ameliorated intestinal radiation injury, made evident by a decrease in myeloperoxidase (MPO) activity, transforming growth factor beta (TGF-ß) immunoreactivity, collagen-I deposition, radiation injury score (RIS) and intestinal serosal thickening. These findings indicate the need for further development of Solulin as a prophylactic and/or therapeutic agent to mitigate radiation-induced intestinal damage.

Brain radiation injury leads to a dose- and time-dependent recruitment of peripheral myeloid cells that depends on CCR2 signaling.

BACKGROUND: Cranial radiotherapy is used to treat tumors of the central nervous system (CNS), as well as non-neoplastic conditions such as arterio-venous malformations; however, its use is limited by the tolerance of adjacent normal CNS tissue, which can lead to devastating long-term sequelae for patients. Despite decades of research, the underlying mechanisms by which radiation induces CNS tissue injury remain unclear. Neuroinflammation and immune cell infiltration are a recognized component of the CNS radiation response; however, the extent and mechanisms by which bone marrow-derived (BMD) immune cells participate in late radiation injury is unknown. Thus, we set out to better characterize the response and tested the hypothesis that C-C chemokine receptor type 2 (CCR2) signaling was required for myeloid cell recruitment following brain irradiation. METHODS: We used young adult C57BL/6 male bone marrow chimeric mice created with donor mice that constitutively express enhanced green fluorescent protein (eGFP). The head was shielded to avoid brain radiation exposure during chimera construction. Radiation dose and time response studies were conducted in wild-type chimeras, and additional experiments were performed with chimeras created using donor marrow from CCR2 deficient, eGFP-expressing mice. Infiltrating eGFP+ cells were identified and quantified using immunofluorescent microscopy. RESULTS: Brain irradiation resulted in a dose- and time-dependent infiltration of BMD immune cells (predominately myeloid) that began at 1 month and persisted until 6 months following ≥15 Gy brain irradiation. Infiltration was limited to areas that were directly exposed to radiation. CCR2 signaling loss resulted in decreased numbers of infiltrating cells at 6 months that appeared to be restricted to cells also expressing major histocompatibility complex class II molecules. CONCLUSIONS: The potential roles played by infiltrating immune cells are of current importance due to increasing interest in immunotherapeutic approaches for cancer treatment and a growing clinical interest in survivorship and quality of life issues. Our findings demonstrate that injury from brain radiation facilitates a dose- and time-dependent recruitment of BMD cells that persists for at least 6 months and, in the case of myeloid cells, is dependent on CCR2 signaling.

Time-course study of different innate immune mediators produced by UV-irradiated skin: comparative effects of short and daily versus a single harmful UV exposure.

The modulatory effects of solar UV radiation on the immune system have been widely studied. As the skin is the main target of UV radiation, our purpose was to compare the impact on skin innate immunity of two contrasting ways to be exposed to sunlight. Hairless mice were UV irradiated with a single high UV dose simulating a harmful exposure, or with repetitive low UV doses simulating short occasional daily exposures. Skin samples were taken at different times after UV irradiation to evaluate skin histology, inflammatory cell recruitment, epidermal T-cell population and the mitochondrial function of epidermal cells. The transcriptional profiles of pro-inflammatory cytokines, chemokines, antimicrobial peptides and Toll-like receptors were evaluated by RT-PCR and ELISA in tissue homogenates. Finally, a lymphangiography was performed to assess modification in the lymphatic vessel system. A single high UV dose produces a deep inflammatory state characterized by the production of pro-inflammatory cytokines and chemokines that, in turn, induces the recruitment of neutrophils and macrophages into the irradiated area. On the other hand, repetitive low UV doses drive the skin to a photo-induced alert state in which there is no sign of inflammation, but the epithelium undergoes changes in thickness, the lymphatic circulation increases, and the transcription of antimicrobial peptides is induced.

[Study of the state of parietal microflora and wall of the large intestine of mice under the influence of anomalous magnetic field].

AIM: Study the possible qualitative and quantitative changes of microbial community of the parietal mucin of the large intestine and the state of the wall of the large intestine in experimental animals underbackground and anomalous influence of geomagnetic field. MATERIALS AND METHODS: CBA mice were put under the influence of anomalous magnetic field comparable to its intensity in Zheleznogorsk (3 Oe) for 1 and 2 weeks. Quantitative and qualitative study of mucous microflora of the large intestine of the mice was performed by bacteriological method. Identification of the microorganisms was performed by microbiological analyzer "Multiskan-Ascent" and commercial test-systems "Lachema-Czech Republic": ENTHEROtest-16, STAPHYtest-16, Streptotest-16, En-COCCUStest-16; for lactobacilli and bifidobacteria identification - API 50 CHL (bioMerieux). Bacteria content in 1 g of material was calculated by the number of microorganism colonies grown. RESULTS: A pattern of changes of mucous microflora of the intestine and the state of the wall of the large intestine of the experimental animals that had been put under the influence of anomalous magnetic field is shown. During evaluation of qualitative and quantitative diversity of microbial community of parietal mucin of the large intestine of the mice under the influence of magnetic field on the background and anomalous levels changes not only in quantity and frequency of detection of obligate, transitory flora but also cell elements of mucous membrane of the wall of the large intestine were established. CONCLUSION: The results of the study allow to make a conclusion about the presence of reactivity of the parietal microflora of the intestine of the mice to the influence of the anomalous magnetic field. This leads to changes in cell elements in the mucous membrane of the wall that manifest by infiltration of the connective tissue stroma by leucocytes and reconstruction of epithelium, that are features of dysbiosis.

The eicosanoid response to high dose UVR exposure of individuals prone and resistant to sunburn.

High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E(2) is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4 h, p < 0.001), neutrophil infiltration (24 h, p = 0.01), epidermal COX-2 (24 h, p < 0.05) and 12-LOX (24 h, p < 0.01), and dermal eNOS (24 h, p < 0.05) proteins, although CD3+ lymphocyte infiltration showed an earlier increase in phototype III/IV (24 h, p < 0.05). Although erythema was equivalent at 72 h in both groups, phototype I/II showed higher PGE(2) accompanied by elevated 15-HETE, and a strong positive correlation was seen between these mediators (n = 18, r = 0.805, p = 0.0001). Hence anti-inflammatory eicosanoid 15-HETE may temper the pro-inflammatory milieu in sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions.

Increased expression of versican in the inflammatory response to UVB- and reactive oxygen species-induced skin tumorigenesis.

Excessive exposure to UV radiation is a major risk factor for developing skin cancer. UV-induced reactive oxygen species (ROS) cause accumulation of DNA damage products such as 8-oxoguanine (8-oxoG) in the skin. We have previously shown that mice lacking the repair enzyme 8-oxoguanine glycosylase (Ogg1 knockout mice) are highly susceptible to skin cancer after long-term UVB exposure. To investigate the genes involved, we performed gene profiling of Ogg1 knockout mouse skin after UVB exposure. Among the up-regulated genes in UVB-treated Ogg1 knockout mice, inflammatory response pathway-related genes were most affected. The Vcan gene, which encodes the large extracellular matrix proteoglycan versican, was continuously up-regulated in UVB-treated Ogg1 knockout mice, suggesting that versican is a mediator of skin cancer development. We examined the expression pattern of versican in skin tumors from wild-type mice and UVB-treated Ogg1 knockout mice, and also analyzed 157 sun-related human skin tumors. Versican was strongly expressed in malignant skin tumors in both mice and humans, and especially in Ogg1 knockout mice. Additionally, infiltrating neutrophils strongly colocalized with versican in UVB-treated Ogg1 knockout mouse skin. These data demonstrate that inflammatory responses, particularly neutrophil infiltration and versican up-regulation, are closely involved in UVB/ROS-induced skin tumorigenesis.

Cranial irradiation leads to acute and persistent neuroinflammation with delayed increases in T-cell infiltration and CD11c expression in C57BL/6 mouse brain.

Radiotherapy is commonly employed to treat cancers of the head and neck and is increasingly used to treat other central nervous system (CNS) disorders. Exceeding the radiation tolerance of normal CNS tissues can result in sequelae contributing to patient morbidity and mortality. Animal studies and clinical experience suggest that neuroinflammation plays a role in the etiology of these effects; however, detailed characterization of this response has been lacking. Therefore, a dose-time investigation of the neuroinflammatory response after single-dose cranial irradiation was performed using C57BL/6 mice. Consistent with previous reports, cranial irradiation resulted in multiphasic inflammatory changes exemplified by increased transcript levels of inflammatory cytokines, along with glial and endothelial cell activation. Cranial irradiation also resulted in acute infiltration of neutrophils and a delayed increase in T cells, MHC II-positive cells, and CD11c-positive cells seen first at 1 month with doses ≥ 15 Gy. CD11c-positive cells were found almost exclusively in white matter and expressed MHC II, suggesting a "mature" dendritic cell phenotype that remained elevated out to 1 year postirradiation. Our results indicate that cranial irradiation leads to persistent neuroinflammatory changes in the C57BL/6 mouse brain that includes unique immunomodulatory cell populations.

Photohardening restores the impaired neutrophil responsiveness to chemoattractants leukotriene B4 and formyl-methionyl-leucyl-phenylalanin in patients with polymorphic light eruption.

A failure to induce immune suppression after UV exposure has been implicated in the pathogenesis of polymorphic light eruption (PLE). This immunological resistance has been linked to an impaired neutrophil infiltration into the skin following UV exposure. Therapeutic photohardening can restore this abnormal neutrophil infiltration in PLE skin and is thought to be responsible for the prophylactic efficacy. The aim of this study was to elucidate the pathogenic mechanism of the described neutrophil deficiency in PLE. Peripheral blood neutrophil responses to the chemoattractants leukotriene B4 (LTB(4)) and formyl-methionyl-leucyl-phenylalanin (fMLP) were investigated in vitro. Samples from 10 patients with PLE before and after 6 weeks of photohardening therapy were assessed. Flow cytometry was used to measure the changes associated with neutrophil activation. We found a significantly reduced neutrophil responsiveness to LTB(4) and fMLP in PLE patients, which was restored to normal levels after phototherapy. Indeed, PLE neutrophil responsiveness to these two chemoattractants after (but not before) phototherapy was similar to that of age- and sex-matched healthy control subjects. This indicates that an abnormal chemotactic potential to neutrophils is a crucial factor in the pathogenesis of PLE. Normalization following photohardening may therefore account for the therapeutic efficacy by restoring UV-induced neutrophil skin infiltration. Our results reveal a completely novel pathogenic mechanism involved in PLE and offer unique targets for therapy.

Photoaged skin: the role of neutrophils, preventive measures, and potential pharmacological targets.

Photoaged skin is characterized by epidermal changes and damaged elastic fiber and collagen fiber networks. Sunburned skin, including minimal asymptomatic ultraviolet (UV)radiation-induced erythema, is characterized by infiltrating neutrophils. Neutrophils are potent cells capable of degrading elastic fibers and collagen fibers and are probably important players in the pathophysiology of photoaging. Therefore,prevention of sunburn and/or prevention of neutrophil influx after exposure to artificial sources of UV radiation appear to be key factors in the prevention of photoaging. The wearing of protective clothing and the use of sunscreens are important preventive measures. Drugs that interfere with the cascade of events that eventually lead to neutrophil influx are potential antiphotoaging agents. In contrast, current and/or future therapies that are accompanied by neutrophil influx, particularly when these therapies are administered or performed repetitively, require a critical review. Here, we discuss clinical and histopathological features of photoaging,the pathophysiology of photoaging, potential pharmacologic targets with respect to infiltrating neutrophils, and some key points in prevention of photoaging and therapy for photoaged skin.

Neutrophil elastase contributes to extracellular matrix damage induced by chronic low-dose UV irradiation in a hairless mouse photoaging model.

BACKGROUND: Repeated exposure to ultraviolet (UV) rays damages skin connective tissue, which is thought to be associated with wrinkle formation. We hypothesized that repeated mild inflammation may cause the connective tissue alterations in photoaging. OBJECTIVE: To clarify the behavior of neutrophils and neutrophil elastase (NE) activity in the photoaging process and to examine the mechanisms of connective tissue damage resulting from NE in photoaging. METHODS: Mouse dorsal skin was irradiated with a suberythemal dose of UVB three times a week. After 5 or 10 weeks of irradiation, neutrophils were investigated by light microscopy and transmission electron microscopy. NE activity was examined by in situ zymography. Activation of proMMP-2 and proMMP-1 by NE both in the purified enzyme and in human skin fibroblast culture was examined by gelatin zymography or immunoblotting respectively. RESULTS: Both neutrophil infiltration and NE activity were elevated in photoaging. Furthermore, activated MMP-2 and MMP-1 were increased by NE treatment in a dose-dependent manner. CONCLUSIONS: In the present study, we demonstrated that neutrophil infiltration and NE activity are elevated in the chronic UVB-irradiated skin of hairless mice and we confirmed the involvement of NE in MMP activation. These data suggest that NE indirectly plays a role in skin photoaging through MMP activation.

Effects of low-level laser therapy on collagen expression and neutrophil infiltrate in 5-fluorouracil-induced oral mucositis in hamsters.

BACKGROUND AND OBJECTIVES: Several studies have suggested that low-level laser therapy (LLLT) can ameliorate oral mucositis; however, the mechanisms involved are not well understood. The aim of this study was to investigate the mechanisms of action of LLLT on chemotherapy-induced oral mucositis, as related to effects on collagen expression and inflammation. MATERIALS AND METHODS: A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil, followed by surface irritation. Animals were randomly allocated into three groups, and treated with an InGaAIP diode laser at a wavelength of 660 nm and output power of 35 or 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. Collagen was qualitatively and quantitatively evaluated after picrosirius staining. The density of the neutrophil infiltrate was also scored. RESULTS: Peak clinical severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW and control groups. The reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a decrease in the number of neutrophils and an increase in the proportion of mature collagen as compared to the other two groups. The total quantity of collagen was significantly higher in the control (no laser) group at the day 11 time-point, as compared to the 35 mW laser group, consistent with a more prolonged inflammatory response in the control group. CONCLUSION: This study supports two mechanisms of action for LLLT in reducing mucositis severity. The increase in collagen organization in response to the 35 mW laser indicates that LLLT promotes wound healing. In addition, LLLT also appears to have an anti-inflammatory effect, as evidenced by the reduction in neutrophil infiltrate.

Development and characterization of a lung-protective method of bone marrow transplantation in the mouse.

Allogeneic bone marrow transplantation is a common method used to study the contribution of myeloid and lymphoid cell populations in murine models of disease. The method requires lethal doses of radiation to ablate the bone marrow. Unintended consequences of radiation include organ injury and inflammatory cell activation. The goal of our study was to determine the degree to which bone marrow transplantation alters lungs and to develop a system to protect the lungs during radiation. C57BL/6 mice were subjected to total body irradiation with 900cGy and then transplanted with bone marrow from green fluorescent protein (GFP) expressing mice. Resultant chimeras exhibited a significant decline in alveolar macrophage numbers within 72h, modest influx of neutrophils in the lungs at 14days, and repopulation of the lungs by alveolar macrophages of bone marrow origin by 28days. Neutrophil influx and alveolar macrophage turnover were prevented when 1cm thick lead shields were used to protect the lungs during radiation, such that 8weeks after transplantation less than 30% of alveolar macrophages were of donor origin. Lung-shielded mice achieved a high level of bone marrow engraftment with greater than 95% of circulating leukocytes expressing GFP. In addition, their response to intratracheal lipopolysaccharide was similar to non-transplanted mice. We describe a model whereby lead shields protect resident cell populations in the lungs from radiation during bone marrow transplantation but permit full bone marrow engraftment. This system may be applicable to other organ systems in which protection from radiation during bone marrow transplantation is desired.