White matter reorganization after surgical resection of brain tumors and vascular malformations.

BACKGROUND AND PURPOSE: Diffusion tensor imaging (DTI) and white matter tractography (WMT) are promising techniques for estimating the course, extent, and connectivity patterns of the white matter (WM) structures in the human brain. In this study, DTI and WMT were used to evaluate WM tract reorganization after the surgical resection of brain tumors and vascular malformations. METHODS: Pre- and postoperative DTI data were obtained in 6 patients undergoing surgical resection of brain lesions. WMT using a tensor deflection algorithm was used to reconstruct WM tracts adjacent to the lesions. Reconstructed tracts included corticospinal tracts, the corona radiata, superior longitudinal and inferior fronto-occipital fasciculi, cingulum bundles, and the corpus callosum. RESULTS: WMT revealed a series of tract alteration patterns including deviation, deformation, infiltration, and apparent tract interruption. In general, the organization of WM tracts appeared more similar to normal anatomy after resection, with either disappearance or reduction of the deviation, deformation, or infiltration present preoperatively. In patients whose lesions were associated with corticospinal tract involvement, the WMT reconstructions showed that the tract was preserved during surgery and improved in position and appearance, and this finding correlated with improvement or preservation of motor function as determined by clinical assessment. CONCLUSION: WMT is useful for appreciating the complex relationships between specific WM structures and the anatomic distortions created by brain lesions. Further studies with intraoperative correlation are necessary to confirm these initial findings and to determine WMT utility for presurgical planning and evaluation of surgical treatments.

Expression of Fas ligand by microglia: possible role in glioma immune evasion.

The immune-privileged status of the central nervous system is thought to limit the application of immunotherapy for treatment of malignant brain tumors. Because the Fas pathway has been proposed to play a role in immune evasion, we examined the effect of tumor environment on the expression of Fas ligand (FasL) in a mouse glioma model. Immunoblotting revealed the expression of membrane-bound FasL to nearly double when murine G26 gliomas were propagated intracranially (IC) as compared to subcutaneously (SC). Further analysis by flow cytometry revealed microglia, which were absent in the SC tumors, to account for half of the FasL expression in the IC tumors. Interestingly, when FasL activity was inhibited in IC tumors, the proportion of tumor-infiltrating leukocytes increased three-fold, reaching the same frequency as the SC tumors. These observations suggest that microglia are a major source of FasL expression in brain tumors and possibly contribute to the local immunosuppressive milieu of malignant gliomas.

Functional MR imaging assessment of a non-responsive brain injured patient.

Functional magnetic resonance imaging (fMRI) was requested to assist in the evaluation of a comatose 38-year-old woman who had sustained multiple cerebral contusions from a motor vehicle accident. Previous electrophysiologic studies suggested absence of thalamocortical processing in response to median nerve stimulation. Whole-brain fMRI was performed utilizing visual, somatosensory, and auditory stimulation paradigms. Results demonstrated intact task-correlated sensory and cognitive blood oxygen level dependent (BOLD) hemodynamic response to stimuli. Electrodiagnostic studies were repeated and evoked potentials indicated supratentorial recovery in the cerebrum. At 3-months post trauma the patient had recovered many cognitive & sensorimotor functions, accurately reflecting the prognostic fMRI evaluation. These results indicate that fMRI examinations may provide a useful evaluation for brain function in non-responsive brain trauma patients.

Reliability of functional MR imaging with word-generation tasks for mapping Broca's area.

BACKGROUND AND PURPOSE: Functional MR (fMR) imaging of word generation has been used to map Broca's area in some patients selected for craniotomy. The purpose of this study was to measure the reliability, precision, and accuracy of word-generation tasks to identify Broca's area. METHODS: The Brodmann areas activated during performance of word-generation tasks were tabulated in 34 consecutive patients referred for fMR imaging mapping of language areas. In patients performing two iterations of the letter word-generation tasks, test-retest reliability was quantified by using the concurrence ratio (CR), or the number of voxels activated by each iteration in proportion to the average number of voxels activated from both iterations of the task. Among patients who also underwent category or antonym word generation or both, the similarity of the activation from each task was assessed with the CR. In patients who underwent electrocortical stimulation (ECS) mapping of speech function during craniotomy while awake, the sites with speech function were compared with the locations of activation found during fMR imaging of word generation. RESULTS: In 31 of 34 patients, activation was identified in the inferior frontal gyri or middle frontal gyri or both in Brodmann areas 9, 44, 45, or 46, unilaterally or bilaterally, with one or more of the tasks. Activation was noted in the same gyri when the patient performed a second iteration of the letter word-generation task or second task. The CR for pixel precision in a single section averaged 49%. In patients who underwent craniotomy while awake, speech areas located with ECS coincided with areas of the brain activated during a word-generation task. CONCLUSION: fMR imaging with word-generation tasks produces technically satisfactory maps of Broca's area, which localize the area accurately and reliably.

Elevation of internal auditory canal pressure by vestibular schwannomas.

OBJECTIVE: The exact mechanism of hearing loss, the most common presenting symptom in patients with vestibular schwannomas, remains unclear. To test whether increased pressure in the internal auditory canal from tumor growth is responsible for this clinical finding, the intracanalicular pressure in patients harboring these tumors was measured. STUDY DESIGN: Prospective study. SETTING: Tertiary referral hospital. PATIENTS: Fifteen consecutive patients undergoing a retrosigmoid approach for resection of vestibular schwannomas were included in the study. INTERVENTION: The intracanalicular pressure in every patient was measured by introducing a pressure microsensor into the internal auditory canal. The pressure readings, which were performed before tumor resection, were then correlated with tumor size and respective preoperative hearing status. RESULTS: Placement of the pressure monitor into the internal auditory canal revealed a biphasic waveform in every patient. Whereas the mean intracanalicular pressure was 20 mm Hg, there was significant variability among patients (range, 1-45 mm Hg). The intracanalicular pressure directly correlated with the amount of tumor in the internal auditory canal (r > 0.63, p < 0.012) but not with the total tumor size (r 0.075). Furthermore, eight patients with class A preoperative hearing (American Academy of Otolaryngology-Head and Neck Surgery classification) had lower intracanalicular pressures than did five patients with class B hearing (16 +/- 5 vs. 28 +/- 4). Although this observation suggested an inverse correlation between the intracanalicular pressure and hearing function, the difference between the two groups was not statistically significant (p = 0.14). CONCLUSION: Pressure on the cochlear nerve as a result of tumor growth in the internal auditory canal may be responsible for hearing loss in patients with vestibular schwannomas. Modification of surgical techniques to address the elevated intracanalicular pressure may be beneficial in improving hearing preservation in these patients.

Role of microglia in glioma biology.

Microglia, a type of differentiated tissue macrophage, are considered to be the most plastic cell population of the central nervous system (CNS). In response to pathological conditions, resting microglia undergo a stereotypic activation process and become capable of phagocytosis, antigen presentation, and lymphocyte activation. Considering their immune effector function, it is not surprising to see microglia accumulation in almost every CNS disease process, including malignant brain tumors or malignant gliomas. Although the function of these cells in CNS inflammatory processes is being studied, their role in malignant glioma biology remains unclear. On one hand, microglia may represent a CNS anti-tumor response, which is inactivated by local secretion of immunosuppressive factors by glioma cells. On the other hand, taking into account that microglia are capable of secreting a variety of immunomodulatory cytokines, it is possible that they are attracted by gliomas to promote tumor growth. A better understanding of microglia-glioma interaction will be helpful in designing novel immune-based therapies against these fatal tumors.

Dexamethasone-induced abolition of the inflammatory response in an experimental glioma model: a flow cytometry study.

OBJECT: Commonly used for management of cerebral edema in patients with brain tumors, steroid medications also have immunosuppressive functions. To characterize the effects of steroids on the central nervous system's response to tumors more clearly, flow cytometry was used to quantify the extent of inflammatory cell infiltration in an immunogenic rat glioma model. METHODS: Freshly prepared 11-day-old intracranial C6 tumors that had been excised from dexamethasone-treated and untreated rats were labeled ex vivo with monoclonal antibodies against CD 11b/c, CD45, and CD8a antigens. The extent of microglia (CD11b/c-highly positive, CD45-slightly positive cell), macrophage (CD11b/c-highly positive, CD45-highly positive cell), lymphocyte (CD11b/c-negative, CD45-highly positive cell), and cytotoxic T-cell (CD8a-positive cell) infiltration into each rat's tumor, tumor periphery, and contralateral tumor-free hemisphere was analyzed using flow cytometry. Microglia and lymphocytes constituted a significant component of infiltrating cells in this model, comprising 23 +/- 3% and 33 +/- 5% of viable cells, respectively. Macrophages, on the other hand, accounted for only 9 +/- 1% of infiltrating cells. Treatment of rats with a 7-day course of low-dose dexamethasone (0.1 mg/kg/day) resulted in a greater than 50% inhibition of microglia (p = 0.03) and lymphocyte (p = 0.001) infiltration into tumors. Increasing the dexamethasone dose to 1 mg/kg/day further abolished lymphocyte infiltration (89% inhibition, p = 0.001) but had no additional inhibitory effect on microglia invasion. Macrophage infiltration of tumors was not inhibited at the dexamethasone doses used in this study (p = 0.42). CONCLUSIONS: Flow cytometry is a valuable technique for characterizing tumor-associated inflammatory cells in gliomas. Even at low doses, dexamethasone was found to inhibit significantly the infiltration of brain tumors by lymphocytes and microglia. These findings should be considered when experimental immunotherapeutic strategies are evaluated for clinical application.

Use of titanium mesh for reconstruction of large anterior cranial base defects.

The authors evaluated the role of titanium mesh used in combination with vascularized pericranium to provide rigid support during reconstruction of anterior skull base defects. Thirteen patients with large anterior skull base defects caused by tumor invasion or traumatic injury involving the cribriform plate, orbital roof, and planum sphenoidale were included in the study. The reconstruction technique involved placement of titanium mesh between two layers of continuous vascularized pericranium. Surgical glue and routine lumbar cerebrospinal fluid (CSF) drainage were not used in any patient. At a mean postoperative follow-up time of 22 months (range 8-39 months), none of the patients had developed infection or meningocele. Postoperative CSF rhinorrhea occurred in two patients with extensive dural defects, which resolved with temporary lumbar drainage. Use of titanium mesh and a two-layer vascularized pericranial graft is a safe, reproducible, and feasible method for reconstructing the anterior skull base. Patients with large dural defects may need temporary CSF diversion to avoid postoperative fistula formation.

Intraoperative sonographic assessment of graft patency during extracranial-intracranial bypass.

Extracranial-intracranial (EC-IC) bypass may be necessary to facilitate treatment of unclippable posterior circulation fusiform aneurysms. Although intraoperative digital subtraction angiography (DSA) allows assessment of graft patency, this technique, because of difficulties inherent in performing selective catheterization and angiography in the operating room, has limitations. Duplex sonography, in contrast, is easily performed, and provides information regarding graft patency and blood flow direction during EC-IC bypass procedures. This latter information proved useful in determining the time of parent artery occlusion after two EC-IC bypass procedures performed for treatment of a fusiform midbasilar artery aneurysm.

Flow cytometric characterization of tumor-associated macrophages in experimental gliomas.

OBJECTIVE: Although microglia have been suggested to be a component of the inflammatory reaction to tumors of the central nervous system, their role in glioma biology remains unknown. One obstacle to studying the function of microglia is the inability to effectively separate them from macrophages. Because flow cytometry can effectively discern immune cells with similar surface antigens, we evaluated its role in characterizing the mononuclear cell infiltration in experimental gliomas. METHODS: Freshly prepared rat C6, 9L, and RG-2 tumor specimens were labeled ex vivo with monoclonal antibodies against CD11b/c, CD45, and CD8a antigens and analyzed by flow cytometry. The extent of microglia (CD11b/c(high), CD45(low)), macrophage (CD11b/c(high), CD45(high)), and lymphocyte (CD11b/c(negative), CD45(high)) infiltration into tumors, tumor periphery, and contralateral tumor-free hemispheres was measured for each glioma type. RESULTS: Microglia, which accounted for 13 to 34% of viable cells, were distributed throughout the central nervous system and were present in the tumors, tumor periphery, and contralateral tumor-free hemispheres. In contrast, macrophages were less prominent within the tumors and tumor periphery (4.2-12%) and were scarce in the contralateral tumor-free hemispheres (0.9-1.1%). Among the tumor types, RG-2 gliomas had the least microglia/macrophage infiltration. The frequency and the distribution pattern of lymphocytes also varied among tumor models. Whereas lymphocytes accounted for more than one-third of the cells in C6 and 9L tumors, they represented only 1% of cells in RG-2 gliomas. CONCLUSION: More abundant than macrophages and scattered throughout the central nervous system, microglia account for a significant component of the inflammatory response to experimental gliomas. A better understanding of microglial function in gliomas may be important in the development of immunotherapy strategies.

Interferon-gamma induces apoptosis and augments the expression of Fas and Fas ligand by microglia in vitro.

Activation of microglia by interferon-gamma (IFN-gamma) has been implicated in a number of central nervous system (CNS) inflammatory disease processes. Because IFN-gamma has also been shown to play a role in programmed cell death, we investigated its cytotoxicity and its effect on the Fas apoptotic pathway in microglia. Flow cytometry was used to quantify the IFN-gamma-mediated apoptotic response and Fas and Fas ligand (FasL) expression in two well-characterized murine microglia cell lines (BV-2 and N9). Nuclear fragmentation, suggestive of apoptosis, was noted within 24 h of incubation of microglia with IFN-gamma (10 U/ml). After a 72-h incubation, almost every BV-2 and N9 microglia, but not GL261 glioma cells, underwent cell death and detached from the culture plates. This cytotoxicity occurred even at low IFN-gamma concentrations (1 U/ml) and was inhibited by BAF, a pan-caspase inhibitor. Incubation of BV-2 and N9 microglia, but not GL261 glioma cells, with IFN-gamma also potentiated the expression of Fas and FasL in a similar dose-response and time-course manner, as seen for the apoptotic response. Whereas Fas expression increased by 100% in both microglia cells, FasL upregulation was more pronounced and increased by as much as 200% in the N9 cells. These findings suggest that in addition to its role as a microglia activator, IFN-gamma may also induce apoptosis of microglia, possibly through simultaneous upregulation of Fas and FasL. Interferon-gamma modulation of the Fas pathway and apoptosis in microglia may be important in the pathogenesis of inflammatory CNS disease processes.

Endoscopic-guided direct endonasal approach for pituitary surgery.

BACKGROUND: Submucosal dissection of the nasal septum is often performed as part of the transseptal approach to the sella. To evaluate whether this submucosal dissection is a necessary component of this operation, we compared the morbidity of a direct transmucosal endonasal approach to that of the transseptal approach in patients undergoing pituitary surgery. METHODS: Forty-one consecutive patients undergoing pituitary surgery from January 1996 to March 1999 were included in this study. The first 21 patients underwent the standard transseptal operation through either a sublabial or columellar incision. The latter 20 patients were operated on through an endoscopically guided, direct endonasal exposure, without any submucosal dissection of the nasal septum. The operative morbidity, the duration of surgery, and the length of hospitalization for each group were compared. RESULTS: The sphenoid sinus exposure obtained through the endonasal route was comparable with the transseptal approach and was adequate for resection of most pituitary tumors. Although the morbidity of the two approaches was similar, patients undergoing the endonasal operation had less postoperative facial pain. Furthermore, the endonasal approach significantly decreased the length of the operation (116 minutes vs. 161 minutes, p = 0.002) and the duration of hospitalization (3.6 vs. 5.1 days, p = 0.003) as compared with the transseptal route. CONCLUSIONS: Morbidity of the endonasal approach to the sphenoid sinus is comparable to that of a conventional transseptal approach. By eliminating the submucosal dissection, the endonasal approach reduces postoperative facial discomfort and decreases length of surgery and hospitalization.

In vitro modulation of microglia motility by glioma cells is mediated by hepatocyte growth factor/scatter factor.

OBJECTIVE: Considered as immune effector cells of the central nervous system, microglia represent a major component of the inflammatory cells found in malignant gliomas. Although their role in brain tumor biology is unclear, accumulation of microglia in malignant brain tumors may be mediated through active secretion of cytokines by glioma cells. Because hepatocyte growth factor/scatter factor (HGF/SF) has been shown to modulate glioma motility through an autocrine mechanism, and because microglia have been reported to express the HGF/SF receptor Met, we hypothesized that microglia recruitment by gliomas may also occur through the secretion of HGF/SF. METHODS: The effect of glioma cells in augmenting BV-2 murine microglia motility was studied by using an in vitro Boyden chamber migration assay. To determine the chemokines involved in microglia migration, neutralizing monoclonal antibodies against monocyte chemotactic protein-1 and HGF/SF were tested. Immunoblotting was used to check for the expression of HGF/SF by glioma cells, and the expression of Met by BV-2 cells was examined by flow cytometry. RESULTS: BV-2 migration was noted within 7 hours of incubation with both human (U251 MG and U373 MG) and murine (GL261) glioma cell lines. This migration corresponded to HGF/SF secretion by glioma cells and was completely inhibited by neutralizing monoclonal antibody against HGF/SF, but not monocyte chemotactic protein-1. Exposure of BV-2 cells to recombinant HGF/SF, but not monocyte chemotactic protein-1, resulted in their migration and down-regulation of Met in a dose-dependent fashion. CONCLUSION: HGF/SF, which plays a role in glioma motility and mitogenesis, may also act as a chemokine for microglia and may be responsible for the microglia infiltration in malignant gliomas. This active recruitment of microglia may play an important role in glioma biology.

Combined radiation and p53 gene therapy of malignant glioma cells.

More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87 MG, p53wt) or mutant (A172 and U373 MG, p53mut) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli beta-galactosidase genes (AdLacZ, control virus) before radiation (0-20 Gy). Changes in p53, p21, and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. Within 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector gene, was overexpressed, and cells were arrested in the G1 phase. Bax expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MG (p53wt) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MG (p53mut) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53mut) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose. Survival of all three cell lines was reduced dramatically after >10 Gy. Although Adp53 transduction significantly reduced the survival of U373 MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line. Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes.

Tumour cell dispersion by the ultrasonic aspirator during brain tumour resection.

Ultrasonic aspirators are commonly used to resect brain tumours because they allow safe, rapid and accurate removal of diseased tissue. Since ultrasonic aspirators generate a spray of aerosolized irrigating fluid around the instrument tip, we questioned whether this spray might contain viable tumours cells that could contribute to intraoperative spread of tumour fragments. To test this hypothesis, we collected the spray produced during the resection of nine brain tumours with an ultrasonic aspirator and semi-quantitatively analysed it for tumour presence. The aerosolized irrigation fluid was found to contain intact tumour cells or clumps of tumour cells in all nine instances, and there was a trend of increasing tumour cell dispersion with increasing ultrasonic aspiration times. Further examination is required to determine if this intraoperative dispersion of apparently viable tumour fragments contributes to local neoplasm recurrence.

Selective intraarterial gene delivery into a canine meningioma.

OBJECT: The goal of this study was to evaluate gene delivery to a benign brain tumor. METHODS: A recombinant adenovirus vector bearing the Escherichia coli beta-galactosidase reporter gene was selectively injected into the vascular supply of a spontaneously occurring canine olfactory groove meningioma. The tumor and a small amount of peritumoral brain tissue were removed 5 days after viral injection and stained with X-Gal to assess gene delivery. The authors noted significant beta-galactosidase gene expression by the tumor, but not by surrounding brain tissue. No obvious viral-related cytotoxicity was noted. CONCLUSIONS: The authors found that meningiomas can be successfully transduced by adenovirus vectors by using endovascular techniques.

Adenovirus-mediated p53 gene delivery potentiates the radiation-induced growth inhibition of experimental brain tumors.

Patients with malignant gliomas continue to have very poor prognosis even after surgical resection, radiation and chemotherapy. Because these tumors often have alterations in the p53 tumor suppressor gene, which plays a key role in the cellular response to DNA damaging agents, we investigated the role of p53 gene therapy in conjunction with ionizing radiation in a rat brain tumor model. Exposure of cultured rat 9L gliosarcoma cells, which contain a mutant p53 gene, to a recombinant adenovirus-vector bearing the wild-type p53 gene (Adp53), induced apoptosis within 24 hours. Although ionizing radiation had no additional effect on apoptosis within this time frame, it caused G1 arrest in non-apoptotic cells after Adp53 therapy. In contrast, wild-type 9L cells demonstrated little G1 arrest after X-irradiation. When animals bearing brain tumors were irradiated after intratumoral Adp53 injections, more than 85% reduction in tumor size was noted. Moreover, the group of rats receiving both radiation and Adp53 therapy had a significant increase in survival as compared to animals receiving either therapy alone. These results support the use of p53 gene therapy as an adjunct to radiation in treatment of malignant brain tumors.

A cost-effectiveness and cost-utility analysis of radiosurgery vs. resection for single-brain metastases.

PURPOSE: The median survival of well-selected patients with single-brain metastases treated with whole-brain irradiation and resection or radiosurgery is comparable, although a randomized trial of these two modalities has not been performed. In this era of cost containment, it is imperative that health-care professionals make fiscally prudent decisions. The present environment necessitates a critical appraisal of apparently equi-efficacious therapeutic modalities, and it is within this context that we present a comparison of the actual costs of resection and radiosurgery for brain metastases. METHODS AND MATERIALS: Survival and quality of life outcome data for radiation alone or with surgery were obtained from two randomized trials, and radiosurgical results were obtained from a multiinstitutional analysis that specifically evaluated patients meeting surgical criteria. Only linear accelerator radiosurgery data were considered. Cost analysis was performed from a societal view point, and the following parameters were evaluated: actual cost, cost ratios, cost effectiveness, incremental cost effectiveness, cost utility, incremental cost utility, and national cost burden. The computerized billing records for all patients undergoing resection or radiosurgery for single-brain metastases from January 1989 to July 1994 were reviewed. A total of 46 resections and 135 radiosurgery procedures were performed. During the same time period, 454 patients underwent whole-brain radiation alone. An analysis of the entire bill was performed for each procedure, and each itemized cost was assigned a proportionate figure. The relative cost ratios of resection and radiosurgery were compared using the Wilcoxon rank sum test. Cost effectiveness of each modality, defined as the cost per year of median survival, was evaluated. Incremental cost effectiveness, defined as the additional cost per year of incremental gain in median survival, compared to the next least expensive modality, was also determined. To calculate the societal or national impact of these practices, the proportion of patients potentially eligible for aggressive management was estimated and the financial impact was determined using various utilization ratios for radiosurgery and surgery. RESULTS: Both resection and radiosurgery yielded superior survival and functional independence, compared to whole brain radiotherapy alone, with minor differences in outcome between the two modalities; resection resulted in a 1.8-fold increase in cost, compared to radiosurgery. The latter modality yielded superior cost outcomes on all measures, even when a sensitivity analysis of up to 50% was performed. A reversal estimate indicated that in order for surgery to yield equal cost effectiveness, its cost would have to decrease by 48% or median survival would have to improve by 108%. The average cost per week of survival was $310 for radiotherapy, $524 for resection plus radiation, and $270 for radiosurgery plus radiation. CONCLUSIONS: For selected patients, aggressive strategies such as resection or radiosurgery are warranted, as they result in improved median survival and functional independence. Radiosurgery appears to be the more cost-effective procedure.

Human neutrophil functions are inhibited in vitro by clinically relevant ethanol concentrations.

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs-chemotaxis and production of reactive oxygen species-and on changes in cytosolic-free calcium ([Ca2+]i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b). fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca2+]i, as assessed by videomicroscopy using the Ca(2+)-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca2+]i levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca2+ influx or in [Ca2+]i.