Enhancement Of Macrophage Migration Research Articles

Phosphoproteomics Reveals Selective Regulation of Signaling Pathways by Lysophosphatidic Acid Species in Macrophages.

Lysophosphatidic acid (LPA) species, prevalent in the tumor microenvironment (TME), adversely impact various cancers. In ovarian cancer, the 18:0 and 20:4 LPA species are selectively associated with shorter relapse-free survival, indicating distinct effects on cellular signaling networks. Macrophages represent a cell type of high relevance in the TME, but the impact of LPA on these cells remains obscure. Here, we uncovered distinct LPA-species-specific responses in human monocyte-derived macrophages through unbiased phosphoproteomics, with 87 and 161 phosphosites upregulated by 20:4 and 18:0 LPA, respectively, and only 24 shared sites. Specificity was even more pronounced for downregulated phosphosites (163 versus 5 sites). Considering the high levels 20:4 LPA in the TME and its selective association with poor survival, this finding may hold significant implications. Pathway analysis pinpointed RHO/RAC1 GTPase signaling as the predominantly impacted target, including AHRGEF and DOCK guanine exchange factors, ARHGAP GTPase activating proteins, and regulatory protein kinases. Consistent with these findings, exposure to 20:4 resulted in strong alterations to the actin filament network and a consequent enhancement of macrophage migration. Moreover, 20:4 LPA induced p38 phosphorylation, a response not mirrored by 18:0 LPA, whereas the pattern for AKT was reversed. Furthermore, RNA profiling identified genes involved in cholesterol/lipid metabolism as selective targets of 20:4 LPA. These findings imply that the two LPA species cooperatively regulate different pathways to support functions essential for pro-tumorigenic macrophages within the TME. These include cellular survival via AKT activation and migration through RHO/RAC1 and p38 signaling.

Leishmania LPG interacts with LRR5/LRR6 of macrophage TLR4 for parasite invasion and impairs the macrophage functions.

Visceral leishmaniasis (VL) is a severe form of leishmaniasis, primarily affecting the poor in developing countries. Although several studies have highlighted the importance of toll-like receptors (TLRs) in the pathophysiology of leishmaniasis, the role of specific TLRs and their binding partners involved in Leishmania donovani uptake are still elusive. To investigate the mechanism of L. donovani entry inside the macrophages, we found that the parasite lipophosphoglycan (LPG) interacted with the macrophage TLR4, leading to parasite uptake without any significant alteration of macrophage cell viability. Increased parasite numbers within macrophages markedly inhibited lipopolysachharide-induced pro-inflammatory cytokines gene expression. Silencing of macrophage-TLR4, or inhibition of parasite-LPG, significantly stemmed parasite infection in macrophages. Interestingly, we observed a significant enhancement of macrophage migration, and generation of reactive oxygen species (ROS) in the parasite-infected TLR4-silenced macrophages, whereas parasite infection in TLR4-overexpressed macrophages exhibited a notable reduction of macrophage migration and ROS generation. Moreover, mutations in the leucine-rich repeats (LRRs), particularly LRR5 and LRR6, significantly prevented TLR4 interaction with LPG, thus inhibiting cellular parasite entry. All these results suggest that parasite LPG recognition by the LRR5 and LRR6 of macrophage-TLR4 facilitated parasite entry, and impaired macrophage functions. Therefore, targeting LRR5/LRR6 interactions with LPG could provide a novel option to prevent VL.

Abstract 4079: IL-6 promotes radiation-induced macrophage infiltration to tumors via upregulation of CCL2 and CCL5

Abstract Background: Radiotherapy is a major treatment modality for non-small cell lung cancer (NSCLC), and it is controversial if radiotherapy contributes to metastasis. We hypothesize that radiotherapy increases lung cancer cell recruitment of macrophage-infiltration, and the infiltrated macrophages may accelerate the metastatic potential of cancer. We examined the role of intracellular IL-6 in NSCLC cells in promoting the recruitment of macrophages after irradiation and explored the molecular mechanisms that govern the increase of macrophages. Methods: By utilizing the IL-6 knocked down (IL-6si) and scramble control (sc) A549 and H157 NSCLC cell lines, we investigated whether the intracellular IL-6 level affected macrophage migration into cancer cells. We also developed orthotopic human xenograft models in nude mice using the luciferase tagged-H157IL-6si and H157sc cells and investigated IL-6 effects on macrophage infiltration to tumor sites after irradiation. Tumors were irradiated and the irradiated mice were divided into two groups: (1) 1st group, the endogenous macrophage-infiltration into tumors was examined; and (2) in the 2nd group, mice were injected with the GFP-labeled THP-1 cells through tail veins after irradiation, and the recruitment of exogenous macrophages (THP-1) to tumor site was investigated 3 days after the injection. We also studied the molecular mechanisms by which IL-6 mediated the increase of macrophage-infiltration upon irradiation. Results: We found a decrease of macrophage recruitment by A549/H157 cells upon radiation when IL-6 was knocked down. Consistent with the in vitro results, we observed the contribution of IL-6 signaling to promote macrophage-infiltration after irradiation in vivo in human tumor xenografts. In the mechanism dissection studies, we found that CCL2 and CCL5 molecules were the critical IL-6 downstream target molecules, which mediated the radiation-induced increase of macrophage infiltration. We confirmed the contribution of these molecules by demonstrating the effects of neutralizing antibodies to these target molecules in reducing the radiation-induced enhancement of macrophage migration to tumors. Conclusion and Impacts: Study results suggest that IL-6 signaling, through the up-regulation of CCL2/CCL5, mediated the increase of macrophage infiltration to NSCLC cells after irradiation. Molecular studies suggest that blocking CCL2/CCL5 pathways can potentially be applied to conventional radiotherapy to intercept the process of radiation-promoted macrophage-infiltration. Citation Format: Xin Wang, Ying Tsai, Kwang-Hsiang Chuang, Peter Keng, Soo Ok Lee, Yuhchyau Chen. IL-6 promotes radiation-induced macrophage infiltration to tumors via upregulation of CCL2 and CCL5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4079.

Cellular sensitization against sperm and seminal antigens in women

In 13 healthy women and 6 virgins the cellular sensitization against sperm and seminal plasma antigens was demonstrated by an indirect lymphokin assay, the leucocyte migration inhibition test (LMI-test) using the following preparations: "washed" spermatozoa, seminal plasma and spermatozoa of the supernatant prepared with the "swim-up" technique. In both groups of women a cellular sensitization against sperm and seminal plasma antigens could be observed. Further, a dose dependent correlation was found in that way, that increasing concentrations of spermatozoa lead to an increased inhibition of macrophage migration. In virgins cellular sensitization against seminal plasma proteins did not differ from non-virgins, only the percentage of significant reactions in the LMI-Test was reduced. As low sperm concentrations (1 million ml-1), which represent best the physiological situation in the uterus, induced an enhanced migrations of macrophages the enhancement of macrophage migration is considered as physiological cellular sensitization of females against sperm-associated antigens.

The Novel Anti-Rheumatic Drug TA-383 Has a Macrophage Migration Enhancing Activity

We have investigated the effect of a novel anti-rheumatic drug, cis-2-(4-chlorophenyl)-4,5-diphenyl-2-imidazoline hydrochloride (TA-383), on macrophages. TA-383 (≥10−9M) significantly stimulated rabbit alveolar macrophage (AM) migration, and its migration-stimulatory activity was more potent than those of L-fucose (5 × 10−3 M), lobenzarit disodium, bucillamine (SA-96) and salazosulfapyridine. In addition, TA-383 enhanced the production and/or release of macrophage migration enhancement factor by rabbit spleen cells. In vivo, TA-383 (0.4 mg/kg, p.o.) has suppressive effects on picryl chloride-induced delayed type hypersensitivity and type II collagen-induced arthritis in mice. These results suggest that the anti-rheumatic activity of TA-383 may be exerted through the dispersion of macrophages from inflammatory sites.

The novel anti-rheumatic drug TA-383 has a macrophage migration enhancing activity.

We have investigated the effect of a novel anti-rheumatic drug, cis-2-(4-chlorophenyl)-4,5-diphenyl-2-imidazoline hydrochloride (TA-383), on macrophages. TA-383 (> or = 10(-9) M) significantly stimulated rabbit alveolar macrophage (AM) migration, and its migration-stimulatory activity was more potent than those of L-fucose (5 x 10(-3) M), lobenzarit disodium, bucillamine (SA-96) and salazosulfapyridine. In addition, TA-383 enhanced the production and/or release of macrophage migration enhancement factor by rabbit spleen cells. In vivo, TA-383 (0.4 mg/kg, p.o.) has suppressive effects on picryl chloride-induced delayed type hypersensitivity and type II collagen-induced arthritis in mice. These results suggest that the anti-rheumatic activity of TA-383 may be exerted through the dispersion of macrophages from inflammatory sites.

Biophysical characterization of macrophage migration enhancement factor (MEF)

Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S 20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 Å. From these measurements the D 20w was calculated to be 7.55 × 10 −7cm 2/sec, the mol. wt was calculated to be 28,000, the frictional ratio (|/| 0) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 20̊ × 160 Å. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100°C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.

Macrophage migration inhibition induced by MDP, LPS, PMA, and MIF/MAF: reversal by macrophage migration enhancement factor (MEF), L-fucose, L-fucosyl BSA, D-mannose, and D-mannosyl BSA.

Our data establish that migration inhibition factor (MIF) and migration enhancement factor (MEF) mutually neutralize the effect of each other in a concentration-dependent manner. The monosaccharides L-fucose and D-mannose were also shown to reverse MIF and additionally to stimulate alveolar macrophage (AM) migration in the absence of MIF. The specific activity of these sugars was increased 200-fold when conjugated to bovine serum albumin (BSA). Macrophage activation is usually observed concurrently with migration inhibition when macrophages are incubated with MIF preparations. Migration inhibition occurred also when AM were incubated in the presence of known metabolic activators (MDP, PMA, and LPS). It was found that L-fucose, D-mannose, L-fucosyl BSA, and D-mannosyl BSA could reverse migration inhibition caused by MIF as well as by these metabolic activators. These observations suggest that reversal of MIF by L-fucose is unexplained solely on the basis that L-fucose is functioning as a competitive inhibitor; instead, they suggest that MEF and the above sugars and their conjugates stimulate AM through a receptor system different from the MIF receptor. These observations support the concept that MEF is an important macrophage modulator in CMI responses.

L-fucose, D-mannose, L-galactose, and their BSA conjugates stimulate macrophage migration.

The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.

Induction of a macrophage migration enhancement factor after desensitization of tuberculin-positive rabbits with purified protein derivative.

The production of a macrophage migration enhancement factor (MEF) has been achieved as a consequence of administering a desensitizing dose of purified protein derivative (PPD) to Mycobacterium bovis BCG-sensitized rabbits. The migration-enhancing effect was first demonstrated when alveolar macrophages (AM) harvested from desensitized rabbits exhibited marked migration stimulation; whereas maximum migration enhancement was observed 8 days after the administration of PPD, migration enhancement of the AM from these rabbits persisted for up to 12 days. Sera from BCG-sensitized, PPD-desensitized animals exhibited a peak of MEF activity 4 days after desensitization. Maximal MEF activity was demonstrated in culture supernatants of nonadherent spleen cells harvested 8 days after the intravenous desensitizing dose of PPD was given. Control spleen cell culture supernatants did not produce detectable MEF. The route of desensitization with PPD was critical. When PPD was administrated intratracheally, MEF activity was not induced. The intravenous administration of BCG after PPD desensitization reversed migration enhancement to strong migration inhibition. Ammonium sulfate fractionation indicated that two fractions contained MEF activity. MEF activity was retained by dialysis membranes with a 15,000-molecular-weight cutoff but passed through dialysis membranes with a 25,000-molecular-weight cutoff. The mixture of migration inhibition factor with MEF-containing supernatants resulted in the mutual cancellation of both activities. These observations suggest that MEF may be a modulator of macrophage effector responses mediated by migration inhibition factor.

Demonstration of a macrophage migration enhancement factor in the sera of young rabbits

Alveolar macrophages (AM), harvested from the lungs of untreated normal young rabbits (New Zealand White) 14 days to 8 weeks of age, exhibited a state of migration stimulation compared to AM from normal adult rabbits (5 to 6 months of age). Migration of AM from normal adult rabbits (New Zealand White) was stimulated 2.0- to 2.5-fold when incubated with sera from 39-to 46-day-old rabbits compared with sera from normal adult rabbits. Furthermore, 4-day spleen cultures obtained from animals 28 to 59 days of age yielded supernatants that also stimulated the migration of adult AM. The spleen cell culture supernatants from 42- to 49-day-old animals had the greatest activity and stimulated the migration of adult AM 2.5- to 3.2-fold compared to the supernatants from adult normal rabbits. The peak production of migration enhancement factor (MEF) by splenic lymphoid cells coincided with the peak activities found in the sera. It was observed that nonadherent peanut agglutinable lymphoid cells produced MEF. When sera or culture supernatants containing MEF were mixed with MIF-containing adult sera or spleen cell culture supernatants, the respective activities were neutralized. The large migrations of normal neonatal AM were diminished by the addition of MIF-containing sera obtained from BCG-sensitized/challenged rabbits. In contrast, AM from BCG-sensitized rabbits, which exhibited a state of reduced migration, were enhanced by MEF-containing sera from untreated young rabbits. Three peaks of MEF activity were detected in Sephadex G-100 column fractionated sera from 42-day-old rabbits having MWs of approximately (Peak I) 80,000, (Peak II) 43,000, and (Peak III) 8000 to 18,000; most of the activity was found in peaks II and III. Two peaks of MEF activity were detected in Sephadex G-100 column-fractionated spleen cell culture supernatants from 42-day-old rabbits having MWs of approximately (Peak I) 35,000 to 43,000 and (Peak II) 10,000 to 14,000; most of the activity was in peak I which corresponds to peak II of the serum fractionation experiment. Collectively, these data indicate that MEF is a lymphokine that could be important in the modulation of cell-mediated immune effector responses.

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): Migration enhancement by lymphokines

The mode of macrophage migration inhibition by lymphokines or a purified macrophage migration inhibitory factor (MIF) was found to differ from that by muramyl dipeptide (MDP) or lipopolysaccharide (LPS). First, a number of spreading macrophages were observed on the border of an aggregated mass of macrophages, the migration of which was inhibited by lymphokines or MIF, whereas few macrophages were spread in the case of MDP or LPS. Second, macrophage migration enhancement occurred at Day 3 in the case of lymphokines or MIF, whereas neither MDP nor LPS caused such an enhancement. These results suggest that macrophage activation by MIF differs from that by MDP or LPS. Also, the enhancement of macrophage migration by MIF at Day 3 is a hitherto unreported property of MIF.

Inhibition and enhancement of macrophage migration in the presence of lymphocytes and seminal fluid of men with fertility disorders.

Macrophage migration inhibition factor (MIF) towards autologous seminal fluid was studied in 29 infertile men, 16 of whom were also examined towards normal seminal fluid. In 10 patients, inhibition of macrophage migration was observed in the presence of 50 microgram of protein of autologous semen sonicates. Nine patients had enhanced macrophage migration in the presence of 5 microgram protein. Macrophage enhancement was more antigen dependent than the migration inhibition. In all patients seminal abnormalities were observed, being milder in the patients whose lymphocytes enhanced macrophage migration.

Acquired cellular resistance, delayed hypersensitivity, and altered macrophage migration in Listeria monocytogenes-infected guinea pigs.

A Listeria monocytogenes infection in guinea pigs was used to study the interrelationship between antigen-induced macrophage migration inhibition, delayed-type hypersensitivity, and acquired cellular resistance. Early after infection (at 2 and 7 days), very significant enhancement of macrophage migration was observed. Migration inhibition was detected beginning on day 14 and was uniformly observed only on day 21 of the infection, after which a shift again to enhancement was seen. The early detection (by day 2) of migration enhancement suggested that this assay may be more sensitive than assessment of delayed type hypersensitivity in vivo, which in this system was first detectable only on day 4. Acquired cellular resistance, as measured by enhanced survival following a high dose challenge with Listeria, was present from day 7 after infection until at least day 60. By splenic clearance studies, however, acquired cellular resistance was present only until day 14 after infection, suggesting that in this system splenic clearance was not a very reliable criterion for measuring acquired cellular resistance.

Studies on contact sensitivity to DNCB in guinea pigs by the macrophage migration test.

Guinea pigs contact-sensitized to 2,4-dinitrochlorobenzene (DNCB) by foot-pad injections or by topical applications of the hapten were studied by the direct macrophage migration test using DNP-guinea pig albumin (DNP-Alb) and DNP-guinea pig skin extract (DNP-SE) as antigens. Specific inhibition of macrophage migration in the presence of either antigen was found in a significant number of animals exhibiting contact sensitivity to DNCB. However, a wide variation in migration indices was observed in both groups of sensitized animals, with some animals showing a significant enhancement of macrophage migration.