Alvar Grönberg

The Human Antimicrobial Peptide LL-37 Suppresses Apoptosis in Keratinocytes

The human cathelicidin antimicrobial peptide LL-37 is involved in various aspects of skin biology, including protection against infection, wound healing, and also in psoriasis. The tight regulation of apoptosis is critical in tissue repair and its deregulation is a part of the psoriasis phenotype. Despite being involved in cell death of several cell types, virtually nothing is known about the function of LL-37 in keratinocyte apoptosis. Here we report that LL-37 peptide protects primary human keratinocytes and HaCaT cells from apoptosis induced by the topoisomerase I inhibitor camptothecin (CAM). In particular, pretreatment with LL-37 significantly decreased caspase-3 activity after CAM-treatment. Expression profiling of keratinocytes treated with LL-37 identified the upregulation of cyclooxygenase-2 (COX-2) expression, a gene implicated in protection from apoptosis. In addition to inducing COX-2 expression, LL-37 stimulated the production of its product, prostaglandin E-2 (PGE-2). Moreover, LL-37 induced the expression of inhibitor of apoptosis-2 (IAP-2), implicated in the COX-2/PGE-2 antiapoptotic pathway. Pretreatment with a selective COX-2 inhibitor abolished the antiapoptotic effect of LL-37 and reduced IAP-2 expression implicating that the antiapoptotic effect of LL-37 in keratinocytes is mediated by a COX-2-dependent mechanism involving IAP-2. Thus, overexpression of LL-37 may contribute to reduced keratinocyte apoptosis in conditions such as psoriasis.

Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers : a randomized, placebo-controlled clinical trial

Venous leg ulcers (VLUs) are one of the most prevalent types of chronic wounds. The aim of this study was to determine the safety and dose–response efficacy of the human synthetic peptide LL‐37 in the treatment of hard‐to‐heal VLUs. This first‐in‐man trial included 34 participants with VLUs and comprised a 3‐week, open‐label, run‐in period on placebo, followed by a 4‐week randomized double‐blind treatment phase with twice weekly applications of LL‐37 (0.5, 1.6, or 3.2u2009mg/mL) or placebo, and a 4‐week follow‐up. The healing rate constants for 0.5 and 1.6u2009mg/mL of LL‐37 were approximately six‐ and threefold higher than for placebo (pu2009=u20090.003 for 0.5u2009mg/mL and pu2009=u20090.088 for 1.6u2009mg/mL). Square‐root transformed wound area data showed improved healing for the 0.5 and 1.6u2009mg/mL dose groups compared with pretreatment values (pu2009<u20090.001 and pu2009=u20090.011, respectively). Consistently, treatment with the two lower doses markedly decreased the mean ulcer area (68% for 0.5u2009mg/mL and 50% for 1.6u2009mg/mL groups). No difference in healing was observed between the groups receiving 3.2u2009mg/mL of LL‐37 and placebo. There were no safety concerns regarding local or systemic adverse events. In conclusion, topical treatment with LL‐37 for chronic leg ulcers was safe and well tolerated with the marked effect on healing predictors at the two lower doses warranting further investigations.

Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18/LL-37 during wounding in human skin in vivo

Please cite this paper as: Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18/LL‐37 during wounding in human skin in vivo. Experimental Dermatology 2009.

Interferon-γ is a strong modulator of NK susceptibility and expression of β2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitts lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitts lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells☆

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.

Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

Background Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes—key cells in wound healing—and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. Methodology/Principal Findings The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe2+, completely prevented mycolactone mediated cytotoxicity. Conclusions/Significance This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

Hybrid resistance against a natural killer (NK) cell-resistant lymphoma (YWA) is mediated by a T cell-dependent mechanism.

Rejection of the Moloney virus-induced YAC lymphoma of strain A origin by semisynegeneic F1 hybrids has previously been shown to correlate with the levels of natural killer (NK) cell activity in the same F1 hybrids against this target cell line in vitro. In the present study, YAC and another Moloney virus-induced lymphoma,, YWA, derived from the A congenic A.SW strain, were tested for F1 hybrid resistance after s.c. inoculation of small numbers of cells into syngeneic and semisyngeneic F1 mice. While YAC cells invariably grew progressively once they formed a palpable tumor, regression of YWA tumors was frequently observed in both susceptible and resistant genotypes. The hybrid resistance pattern for YAC and YWA differed in one important respect: outcross of the syngeneic host to the A-congenic A.BY strain introduced a strong H-2b-associated resistance factor against YWA, but not against YAC. Compared to YAC, which is highly NK-sensitive and rapidly eliminated from mice with high NK activity, YWA was insensitive to NK-mediated lysis in vitro and [125I] UdR-labelled YWA cells were not eliminated more efficiently from the highly resistant (A.SW X A.BY) F1 then from the parental strain in short-term (4-18h) in vivo rejection assays. It was therefore concluded that the H-2b-associated resistance against YWA was independent of NK cells or other rapidly acting effector mechanisms. Moreover, thymectomy, followed by irradiation and fetal liver reconstitution, completely abolished the resistance against YWA but left the resistance against YAC virtually intact. These data suggest that two lymphomas induced by the same agent can be rejected by different effectors. The NK-resistant YWA lymphoma is rejected by a T-dependent mechanism, while the resistance against the inoculation of the highly NK-sensitive YAC line is T-independent and, in all probability, mediated by NK cells.

Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules

Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear.

NON-NK LEUKOCYTES DEMONSTRATE NK-PATTERNED BINDING

Publisher Summary This chapter discusses the nonkiller cell (NK) leukocytes, which demonstrate NK-patterned binding. In a study described in the chapter, a panel of mouse and human tumor targets with different level of sensitivity to mouse NK activity was used. The levels of NK sensitivity on an individual tumor were also varied by selecting NK resistant variants from the NK sensitive YAC-1 lymphoma. These variants had a decreased NK sensitivity as lower % TBC by nylon passed spleen cells, adherent peritoneal cells and adult and newborn thermocytes. The fact that %TBC correlates weakly with lysis but strongly with the competing ability of these tumor cells suggests that the target binding assay is an adequate measurement of selectivity in the NK system. The finding also illustrates that an interpretation of NK cell frequency based upon % TBC is misleading unless purified NK populations are used. The findings that NK-patterned binding occurs with leukocytes devoid of NK lytic activity shows that binding and lysis are separately controlled events. As there is no evidence that thymocytes have pre-NK cells, the NK patterned binding is not restricted to NK cells. It is probable that qualitative difference exist among the binding of lytically active and inactive effector cells.

Decreased sensitivity of immunoselected and NK-selected YAC lymphoma sublines to hybrid resistance in vivo

Two sublines of the Moloney lymphoma YAC, selected by alternating in vitro exposure to anti-MCSA + complement and in vivo passage in preimmunized hosts, had a decreased or undetectable expression of MCSA. These immunoselected sublines were compared with the original YAC line with regard to their ability to grow in a panel of semisyngeneic F1 hybrids. Natural hybrid resistance to YAC, previously found to be mediated by NK cells, affected the immunoselected sublines to a much smaller extent. This was further corroborated by the fact that the same sublines showed a decreased sensitivity to the in vitro lysis by NK cells from the same hybrid genotypes. Another set of YAC variants were produced by repeated in vitro exposure to NK cells and intermittent passage in highly NK-active F1 hosts. These NK-selected sublines showed a permanently decreased sensitivity to NK lysis after 8-10 selections. When compared for in vivo growth with the parental YAC-1 tissue culture line in a spectrum of relatively resistant F1 hybrids, they had an increased frequency of takes. This is in line with recent findings which show a relationship between the target site for natural antibodies and anti-MCSA on the one hand, and between the natural antibody-binding site and the NK target site on the other.