Defective Neutrophil Research Articles

Phagocytosis of Apoptotic Neutrophils Regulates Granulopoiesis via IL-23 and IL-17

Homeostatic regulation of neutrophil production is thought to match neutrophil elimination to maintain approximately constant numbers in the blood. Here, we show that IL-17, a cytokine that regulates granulopoiesis through G-CSF, is made by gammadelta T cells and unconventional alphabeta T cells. These neutrophil-regulatory T cells (Tn) are expanded in mice that lack leukocyte adhesion molecules, which have neutrophilia and defective neutrophil trafficking. Normal neutrophils migrate to tissues, where they become apoptotic and are phagocytosed by macrophages and dendritic cells. This curbs phagocyte secretion of IL-23, a cytokine controlling IL-17 production by Tn cells. Adoptive transfer of wild-type, but not adhesion molecule-deficient, neutrophils into mice deficient in beta2 integrins transiently decreases neutrophilia and reduces levels of serum IL-17. Antibody blockade of the p40 subunit of IL-23 reduces neutrophil numbers in wild-type mice. These findings identify a major homeostatic mechanism for the regulation of neutrophil production in vivo.

Primary and secondary autoimmune neutropenia

Antineutrophil antibodies are well recognized causes of neutropenia, producing both quantitative and qualitative defects in neutrophils and increased risk for infection. In primary autoimmune neutropenia (AIN) of infancy, a moderate to severe neutropenia is the sole abnormality; it is rarely associated with serious infections and exhibits a self-limited course. Chronic idiopathic neutropenia of adults is characterized by occurrence in late childhood or adulthood, greater prevalence among females than among males, and rare spontaneous remission. Secondary AIN is more commonly seen in adults and underlying causes include collagen disorders, drugs, viruses and lymphoproliferative disorders. In most patients with AIN, antibodies recognize antigens located on the IgG Fc receptor type 3b but other target antigens have been recently identified in secondary AIN. Granulocyte colony-stimulating factor is a proven treatment in patients with AIN of all types and is now preferred to other possible therapies.

Primary immune deficiencies − improving our understanding of their role in immunological disease

Primary immune deficiencies − improving our understanding of their role in immunological disease

Rac2-Deficient Murine Macrophages Have Selective Defects in Superoxide Production and Phagocytosis of Opsonized Particles

The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific Rac2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of Rac2-null murine macrophages were characterized to examine whether Rac2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and Rac2 are present in similar amounts, Rac1 was approximately 4-fold more abundant than Rac2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of Rac2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in rac2(-/-) mice. FcgammaR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in Rac2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcgammaR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in rac2(-/-) macrophages. Macrophage morphology was also similar in wild-type and Rac2-null cells, as was actin polymerization induced by FcgammaR-mediated phagocytosis or M-CSF. Hence, Rac2-null macrophages have selective defects paralleling many of the observed functional defects in Rac2-null neutrophils. These results provide genetic evidence that although Rac2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.

MD-2 is required for the full responsiveness of mast cells to LPS but not to PGN

To address the role played by MD-2 in mast cell recognition of LPS, we examined bone marrow-derived mast cells (BMMCs) from MD-2 gene-targeted mice. BMMCs from MD-2−/− mice showed impaired cytokine production (TNF-α, IL-6, IL-13, and IL-1β) in response to LPS from Escherichia coli, but not to peptidoglycan (PGN) from Staphylococcus aureus. In a mast cell-dependent acute septic model, MD-2 deficiency of mast cell resulted in significantly higher mortality due to defective neutrophil recruitment and the production of cytokines in the peritoneal cavity, which was similar to mice with TLR4-deficient mast cells. The TLR2-dependent activation of skin mast cells by PGN was not altered by the absence of MD-2 in vivo. Collectively, MD-2 is essential for the recognition of LPS by TLR4 but not for that of PGN by TLR2 of mast cells.

Leptin as a uremic toxin interferes with neutrophil chemotaxis.

Leptin is a pleiotropic molecule involved in energy homeostasis, hematopoiesis, inflammation, and immunity. Hypoleptinemia characterizing starvation has been strictly related to increased susceptibility to infection secondary to malnutrition. Nevertheless, ESRD is characterized by high susceptibility to bacterial infection despite hyperleptinemia. Defects in neutrophils play a crucial role in the infectious morbidity, and several uremic toxins that are capable of depressing neutrophil functions have been identified. Only a few and contrasting reports about leptin and neutrophils are available. This study provides evidence that leptin inhibits neutrophil migration in response to classical chemoattractants. Moreover, serum from patients with ESRD inhibits migration of normal neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine with a strict correlation between serum leptin levels and serum ability to suppress neutrophil locomotion. Finally, the serum inhibitory activity can be effectively prevented by immune depletion of leptin. The results also show, however, that leptin by itself is endowed with chemotactic activity toward neutrophils. The two activities-inhibition of the cell response to chemokines and stimulation of neutrophil migration-could be detected at similar concentrations. On the contrary, neutrophils exposed to leptin did not display detectable [Ca(2+)](i) mobilization, oxidant production, or beta(2)-integrin upregulation. The results demonstrate that leptin is a pure chemoattractant devoid of secretagogue properties that are capable of inhibiting neutrophil chemotaxis to classical neutrophilic chemoattractants. Taking into account the crucial role of neutrophils in host defense, the leptin-mediated ability of ERSD serum to inhibit neutrophil chemotaxis appears as a potential mechanism that contributes to the establishment of infections in ERSD.

Annexins – unique membrane binding proteins with diverse functions

Annexins are a well-known multigene family of Ca(2+)-regulated phospholipid-binding and membrane-binding proteins. Recent work employing annexin-knockdown or - knockout models has provided new insights into the biological functions of different annexin proteins. Transient annexin depletion by RNA interference and the expression of dominant-negative mutant proteins has revealed roles for the proteins in membrane processes ranging from the control of membrane structure to certain membrane transport phenomena. Although such functions correlate well with the ability of annexins to interact with cellular membranes in a reversible and regulated manner, some activities are membrane independent, probably because annexins can also engage in specific protein-protein interactions. Among other things, this is evident in annexin A1- and A2-knockout mice, which show impaired regulation of neutrophil extravasation and defects in plasmin generation, respectively.

Defective neutrophil oxidative burst in preterm newborns on exposure to coagulase-negative staphylococci.

The neutrophil oxidative burst is a product of the regulated assembly of the multicomponent oxidase enzyme. Our aim was to compare the oxidative burst in term (n = 10) and preterm newborns <31 wk gestational age (n = 10) after stimulation with coagulase-negative staphylococci in vitro. Strains of Streptococcus epidermidis with different invasive and slime-producing properties, one strain of S. haemolyticus, and one strain of group B-streptococcus were investigated. A whole-blood flow cytometric assay using the oxidation of hydroethidine to ethidium bromide was used. The oxidative activity in unstimulated neutrophil granulocytes [polymorphonuclear leukocytes (PMNLs)] was similar in term and preterm newborns, but the preterm newborns showed a significantly lower capacity to up-regulate the oxidative burst intensity after bacterial stimulation (p = 0.004). In the term but not in the preterm group, the oxidative burst intensity after bacterial stimulation correlated with the baseline oxidative burst intensity. After bacterial stimulation, there was a trend toward a greater percentage of activated neutrophils in the term group than in the preterm group, but the difference was less pronounced than that in oxidative burst intensity. Significant differences in oxidative burst response to different bacterial strains were observed (p < 0.001), but the differences could not be correlated exclusively to invasive capacity or slime-producing properties. It is concluded that the baseline oxidative activity is similar in term and preterm PMNLs but that preterm PMNLs have a decreased capacity to increase the oxidative burst in response to bacterial stimulation.

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia.

The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K-Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A.

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

The chemotaxis defect of Shwachman-Diamond Syndrome leukocytes.

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.

Gene expression profiling provides insight into the pathophysiology of chronic granulomatous disease.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcgammaR1, FcalphaR, CD120b, TLR5, IL-4R, CCR1, p47(phox), p40(phox), IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Adjunctive granulocyte colony-stimulating factor for treatment of septic shock due to melioidosis.

Melioidosis, caused by the intracellular pathogen Burkholderia pseudomallei, is endemic in northern Australia and Southeast Asia. Risk factors for this infection have also been associated with functional neutrophil defects. Because of this, granulocyte colony-stimulating factor (G-CSF) was adopted for use in patients with septic shock due to melioidosis in December 1998. We compared the mortality rates from before and after the introduction of G-CSF therapy at the Royal Darwin Hospital (Darwin, Australia) during the period of 1989-2002. The mortality rate decreased from 95% to 10% after the introduction of G-CSF. Risk factors, the duration of illness before presentation, and the severity of illness were similar in both groups. A smaller decrease in mortality among patients in the intensive care unit who did not have melioidosis was observed, suggesting that other changes in management did not account for the magnitude of the benefit seen. We conclude that G-CSF may have contributed to the reduction in the mortality rate among patients with septic shock due to melioidosis.

Overproduction of neutrophil radical oxygen species correlates with negative symptoms in schizophrenic patients: parallel studies on neutrophil chemotaxis, superoxide production and bactericidal activity

Defective neutrophil function in schizophrenic patients has recently been reported. There are several lines of evidence to support the contribution of oxygen free radicals in schizophrenia, including increased lipid peroxidation, fatty acids and alterations in blood levels of anti-oxidant enzymes. Eighteen schizophrenic patients (DSM-IV) and 15 healthy controls were studied. Neutrophil chemotaxis, superoxide production and bactericidal activity were investigated. A statistically significant increase of superoxide anion release was found in schizophrenic patients compared with controls (mean±S.E.M., patients: 6.89±0.30 nmol O 2 −/10 6 cells/min, controls: 5.13±0.55 nmol O 2 −/10 6 cells/min). Moreover, a significant positive correlation between superoxide production and negative symptoms as assessed by the Positive and Negative Syndrome Scale was demonstrated. No differences were detected in chemotaxis and phagocytosis between schizophrenic patients and healthy controls. The present findings of a positive correlation between superoxide generation and negative symptoms in schizophrenic patients support the hypothesis that superoxide anion may participate in the pathogenesis of schizophrenia, as an excess of free radicals could contribute to the deterioration phase of the disease. Further studies are required to establish the role of oxidative stress in the ethiopathogenesis of schizophrenia.

Salmonella as a Causative Organism of Various Infections in Patients with Sickle Cell Disease

Patients with sickle cell disease (SCD) are susceptible to a variety of bacterial infections, including infections caused by Salmonella species. We sought to identify the most common infections caused by Salmonella spp. in SCD patients at our hospital, and to determine their mortality rates and susceptibility to commonly used antimicrobial agents. We conducted a retrospective review of all Salmonella infections in SCD patients admitted to Qatif Central Hospital during the last 11 years. During the 11 years of the study, 331 episodes of Salmonella infection occurred in 319 patients. The incidence of Salmonella infection in SCD patients was 11.5% compared to only 0.65% in the general hospital population. The commonest sites of Salmonella infection were blood (42% of isolates), followed by gastrointestinal tract (23.5%) and the skeletal system (22.5%). Salmonella infection was commonly seen in patients less than 15 years of age. The case fatality rate in patients with Salmonella sepsis was 2.2% (3/139), but there were no deaths associated with other types of Salmonella infections. Ampicillin and cotrimoxazole resistance occurred in 29% and 24% of the isolates, respectively, but only 0.6% were resistant to ceftriaxone and 1.8% had intermediate resistance to ciprofloxacin. Salmonella is a common cause of sepsis and osteomyelitis in patients with SCD in our locality. Empirical antimicrobial therapy should cover the likely pathogens, including Salmonella species.

The investigation of the ultrastructural neutrophil changes in alloxan-induced diabetes in rats: response to a chemotactic challenge.

Experimental diabetes is one of the most popular conditions in which to study the relation between neutrophil leukocyte activity and periodontal destruction. The aetiology of neutrophil dysfunction in the gingival tissue associated with diabetes has yet to be clarified. Diabetes in rats decreases neutrophil chemotactic activity in proportion to the severity of this systemic disorder. The present study was carried out to evaluate the relationship between the severity of diabetes and the neutrophil response to two chemotactic agents, and to correlate the observed neutrophil defects with the degree of diabetes. In this study two chemotactic agents, casein (0.2 microl, 2 mg ml(-1)) or N-formylmethionylleucylphenylalanine (FMLP; 0.2 microl, 10(-4) M), were placed into the gingival crevices of alloxan-induced diabetic rats. Gingival biopsies were taken 15 min later and then at 5-min intervals up to 45 min and investigated by electron microscopy. Adherence and migration were observed in the rats with moderate diabetes 30 min after the application of casein. There was chemotaxis after 35 min of administration of the peptide FMLP. By 40 min neutrophils with pyknotic nuclei were observed. At 45 min neutrophils with a decreased number of granules were present. As the severity of the diabetes increased, the neutrophils degenerated and were structurally distorted. In the rats which had alloxan-induced diabetes there was abnormal periodontal damage. This damage is thought to be related to dysfunctional neutrophils. These findings many contribute to an answer to the following question: why is there an apparent variability in the susceptibility of periodontal breakdown in diabetics?

Effect of anatomical distribution of mast cells on their defense function against bacterial infections: demonstration using partially mast cell-deficient tg/tg mice.

Mast cells were depleted in the peritoneal cavity of WBB6F1-tg/tg mice that did not express a transcription factor, MITF. When acute bacterial peritonitis was induced in WBB6F1-+/+, WBB6F1-W/W v, and WBB6F1-tg/tg mice, the proportion of surviving WBB6F1-+/+ mice was significantly higher than that of surviving WBB6F1-W/W v or WBB6F1-tg/tg mice. The poor survival of WBB6F1-W/W v and WBB6F1-tg/tg mice was attributed to the deficient influx of neutrophils into the peritoneal cavity. The injection of cultured mast cells (CMCs) derived from WBB6F1-+/+ mice normalized the neutrophil influx and reduced survival rate in WBB6F1-W/W v mice, but not in WBB6F1-tg/tg mice. This was not attributable to a defect of neutrophils because injection of TNF-α increased the neutrophil influx and survival rate in both WBB6F1-W/W v and WBB6F1-tg/tg mice. Although WBB6F1-+/+ CMCs injection normalized the number of mast cells in both the peritoneal cavity and mesentery of WBB6F1-W/W v mice, it normalized the number of mast cells only in the peritoneal cavity of WBB6F1-tg/tg mice. Mast cells within the mesentery or mast cells in the vicinity of blood vessels appeared to play an important role against the acute bacterial peritonitis. WBB6F1-tg/tg mice may be useful for studying the effect of anatomical distribution of mast cells on their antiseptic function.

Defective production of IL-18 and IL-12 by cord blood mononuclear cells influences the T helper-1 interferon gamma response to group B Streptococci.

Human neonates are uniquely susceptible to group B streptococcal (GBS) infections. We have shown that neonatal mixed mononuclear cells have a deficiency in the production of the T helper-1 (Th-1) cytokine, interferon gamma (IFN-gamma), and that incubation of neonatal neutrophils with recombinant IFN-gamma corrects these neutrophil defects. IL-12 and the more recently described IL-18 are also Th-1 type cytokines that are able to induce the production of IFN-gamma in the presence of bacteria and bacterial products. We examine the ability of GBS to induce the production of IFN-gamma, IL-18, and IL-12 by cord blood mixed mononuclear cells and compared these results with the IFN-gamma, IL-18, and IL-12 response of mixed mononuclear cells from adult blood. We demonstrate that cord blood mixed mononuclear cells produced significantly less IFN-gamma, IL-18, and IL-12 in response to GBS compared with mixed mononuclear cells from adults. Cord blood mixed mononuclear cells' production of IFN-gamma is enhanced by added recombinant IL-18 and IL-12. The maximal cord blood cell production of IFN-gamma, in response to GBS, is achieved by priming the cells with both IL-18 and IL-12. We conclude that neonatal mixed mononuclear cells exhibit deficiencies in three main Th-1 type cytokine responses, IFN-gamma, IL-12, and IL-18. This combined Th-1 type cytokine deficiency may contribute to the enhanced susceptibility of the human neonate to GBS and other microbial infections.

An Open-Label Pilot Study of Granulocyte Colony-Stimulating Factor for the Treatment of Severe Endoscopic Postoperative Recurrence in Crohn’s Disease

Background/Aim: Recombinant human granulocyte colony-stimulating factor (rhG-CSF) promoted healing of Crohn’s disease (CD)-like intestinal lesions in chronic granulomatous disease and glycogen storage disease Ib, both characterized by defective neutrophil functions. We performed a prospective, open-label pilot study with rhG-CSF for the treatment of CD. Patients andMethods: Five patients with clinically inactive CD, but with severe endoscopic ileitis within 1 year after intestinal resection and ileocolonic anastomosis, received 300 µg of rhG-CSF (Filgrastim; Neupogen^®) subcutaneously, three times weekly for a total of 12 weeks. Safety was evaluated by assessment of clinical and laboratory data and disease activity. The primary parameter of efficacy was complete mucosal healing, as defined by the Rutgeerts score. Anti-inflammatory mediators were repeatedly measured during treatment. Results: All patients completed the protocol in clinical remission. In 1 subject transient headache resolved after halving the rhG-CSF dosage. Complete mucosal healing was observed in 2 patients: in 1 patient after 12 weeks of therapy and in 1 patient 9 months after treatment cessation. In a single patient, closure of an anovaginal and of a perianal fistula was noted. Neutrophil counts and interleukin-1 receptor antagonist and soluble tumor necrosis factor receptor p55 and p75 levels were found to be increased during drug administration. Conclusion: rhG-CSF seems to be safe, well tolerated, and might provide efficacy in CD.

Comparison of L-Selectin and CD11b on Neutrophils of Adults and Neonates during the First Month of Life

The newborn infant is particularly susceptible to infection in the first weeks of life and this may be, in part, related to functional impairment of neonatal neutrophils in regard to adherence, chemotaxis, and migration. Differences in expression of the neutrophil adherence molecules, l-selectin and CD11b/CD18 (Mac-1), have been previously demonstrated in cord blood and in very young infants (≤ 48 h) compared with adults, but it is unknown for how long these differences persist. We measured surface expression of neutrophil l-selectin and CD11b using flow cytometry in healthy term human newborns from 24 h through 4 wk of age. We also measured levels of soluble l-selectin using an ELISA in neonates though the age of 4 wk. Compared with adults, neonates expressed significantly less l-selectin on resting neutrophils through 4 wk of age, with the lowest levels being at 24 h. Expression of l-selectin on the neutrophil after activation with N-formyl-methionyl-leucyl-phenylalanine was less in the neonate than in the adult (p < 0.05) for the first week of life. Soluble l-selectin showed a steady increase with age in the neonates. Soluble l-selectin was significantly lower in the 24-h neonate compared with the adult and was higher in the 4-wk neonate compared with the adult. CD11b expression was similar between neonates and adults on unstimulated neutrophils, but upon activation, the neonatal neutrophil demonstrated significantly lower up-regulation of CD11b on the neutrophil surface through 4 wk of age compared with adults (p < 0.05 for all ages except 1 wk). These findings that differences in expression of l-selectin and CD11b from that in adults persist throughout the neonatal period provide further evidence that these differences may play a role in the neutrophil defects observed during the neonatal period.