Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 m² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 m² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Estradiol Is a Critical Mediator of Macrophage-Nerve Cross Talk in Peritoneal Endometriosis

Endometriosis occurs in approximately 10% of women and is associated with persistent pelvic pain. It is defined by the presence of endometrial tissue (lesions) outside the uterus, most commonly on the peritoneum. Peripheral neuroinflammation, a process characterized by the infiltration of nerve fibers and macrophages into lesions, plays a pivotal role in endometriosis-associated pain. Our objective was to determine the role of estradiol (E2) in regulating the interaction between macrophages and nerves in peritoneal endometriosis. By using human tissues and a mouse model of endometriosis, we demonstrate that macrophages in lesions recovered from women and mice are immunopositive for estrogen receptor β, with up to 20% being estrogen receptor α positive. In mice, treatment with E2 increased the number of macrophages in lesions as well as concentrations of mRNAs encoded by Csf1, Nt3, and the tyrosine kinase neurotrophin receptor, TrkB. By using in vitro models, we determined that the treatment of rat dorsal root ganglia neurons with E2 increased mRNA concentrations of the chemokine C-C motif ligand 2 that stimulated migration of colony-stimulating factor 1–differentiated macrophages. Conversely, incubation of colony-stimulating factor 1 macrophages with E2 increased concentrations of brain-derived neurotrophic factor and neurotrophin 3, which stimulated neurite outgrowth from ganglia explants. In summary, we demonstrate a key role for E2 in stimulating macrophage-nerve interactions, providing novel evidence that endometriosis is an estrogen-dependent neuroinflammatory disorder.Endometriosis affects 10% of reproductive age women and is associated with persistent pelvic pain.¹ It is defined by the presence of endometrial-like tissue (lesions) found outside the uterus, most commonly on the peritoneum. The mechanisms underlying endometriosis-associated pain are poorly understood, but it has been postulated that estrogen-dependent neuroinflammation may be involved.² Notably, the presence of endometrial tissue fragments on the peritoneum elicits an immune response, including recruitment of macrophages,³ blood vessels, and nerve fibers into the resultant lesions.^4,5 Within the lesions, CD68⁺ macrophages have been detected in close association with nerve fibers.⁶Studies investigating macrophage activation and recruitment have revealed that endometriosis-associated macrophages exhibit a phenotype consistent with the alternative end of the macrophage activation spectrum.^7,8 In a mouse model of endometriosis that included cell transfer of polarized macrophages, Bacci et al⁷ reported that mice injected with proinflammatory macrophages [macrophage (interferon γ)] developed microscopic lesions, but those injected with alternatively activated macrophages [macrophage (IL-4)] developed larger lesions with a well-developed vasculature. Our studies in a mouse model of endometriosis have revealed that macrophages resident in peritoneal lesions can originate from both the peritoneum and the endometrium.⁹Sensory C, sensory Aδ, cholinergic, and adrenergic nerve fibers have been identified within lesions,^10,11 with greater nerve fiber density in areas that exhibit high macrophage density.⁶ Studies in zebrafish have shown that macrophages will migrate toward damaged peripheral nerves,¹² consistent with a role for neuron-derived factors in immune-nerve cross talk.Endometriosis lesions have an estrogen-rich microenvironment associated with enhanced expression of biosynthetic enzymes, including aromatase.¹³ It is well established that estrogen action can be mediated by estrogen receptors α (ERα) and β (ERβ), both widely expressed are the human endometrium.¹⁴ Notably, a proportion of the soma of afferent nerve fibers innervating the uterus and peritoneum is reported to express one or both ERs.¹⁵ Although some studies have analyzed the expression of ERs in macrophages isolated from the peritoneal fluid of women with endometriosis,^16,17 expression of ERs in lesion-resident macrophages has not yet been determined.Our objective was to determine whether estradiol (E2) plays a role in the regulation of macrophage-nerve cross talk in endometriosis by exploring both the expression of ERs in human tissue samples and the impact of E2 on nerves and macrophages using in vitro and in vivo models.     

Pneumocystis carinii stimulates in vitro production of tumor necrosis factor-α by human macrophages

The ability of Pneumocystis carinii to induce tumor necrosis factor (TNF)- release by monocytes/macrophages from immunocompetent humans was investigated. Monocytes and monocyte-derived macrophages from healthy individuals produced an increased amount of TNF- when exposed to P. carinii cysts obtained from rats with steroid-induced pneumocystosis. The cysts induced increased TNF- production in a dose-dependent manner; baseline TNF- production was restored after addition of an anti-P. carinii hyperimmune serum. Kinetics experiments showed that the secretion of TNF- occurs early and reachs a maximal peak after 8 h. Since TNF- is directly lethal to P. carinii in vitro, it is suggested that the production of this cytokine in response to the cysts may be one of the mechanisms for the control of this parasitic infection.     

Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8⁺ immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8⁺ T cells does not require their production of interferon-γ, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8⁺ T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting many warm-blooded mammals including humans. Acute infection is characterized by proliferation of tachyzoites and is known to cause various diseases including lymphadenitis and congenital infection of fetuses.¹ Interferon (IFN)-γ-mediated immune responses limit proliferation of tachyzoites, but the parasite establishes a chronic infection by forming cysts, which can contain hundreds to thousands of bradyzoites, primarily in the brain. Chronic infection with T. gondii is one of the most common parasitic infections in humans. It is estimated that 5 × 10⁸ people worldwide are chronically infected with this parasite.² The tissue cysts remain largely quiescent for the life of the host, but can reactivate and cause life-threatening toxoplasmic encephalitis in immunocompromised patients, such as those with AIDS, neoplastic diseases and organ transplants.^3,4 In immunocompetent individuals, recent studies suggested that T. gondii is an important cause of cryptogenic epilepsy,^5,6 and is likely involved in the etiology of schizophrenia.^7,8 The tissue cyst is not affected by any of the current drug treatments and it has been generally regarded as untouchable. However, the immune responses against T. gondii cysts remain largely unexplored.Resistance to T. gondii is under genetic control in humans^9,10 and mice.^11,12 BALB/c mice are genetically resistant and have only small numbers of cysts in their brains at 2 to 3 months after infection.^11,12 These mice may be able to prevent formation of cysts by efficiently controlling proliferation of tachyzoites during the acute stage of infection. However, it is also possible that the immune system of these animals has the capability to recognize T. gondii cysts and eliminate them from their brains. To examine whether immune cells have an activity to remove cysts that have already been formed in the brain, we adoptively transferred immune cells obtained from chronically infected BALB/c mice into infected, sulfadiazine-treated athymic nude or severe combined immunodeficient (SCID) mice, both of which lack T cells and developed large numbers of cysts in their brains. We present evidence for a potent capability of CD8⁺ immune T cells to eliminate T. gondii cysts from the brains through their perforin-mediated activity.     

Membrane specializations in the first optic neuropil of the housefly,Musca domestica L. I. Junctions between neurons

Summary Thin section and freeze-fracture replicas of the first optic neuropil (lamina ganglionaris) of the flyMusca were studied to determine the types, extent and location of membrane specializations between neurons. Five junctional types are found, exclusive of chemical synapses. These are gap, tight and septate junctions, close appositions between retinular (R) axons and capitate projections (in which an epithelial glial cell invaginates into an R axon). Junctional types and their cellular associations follow: gap junctions, between lamina (L) interneurons, L1–L2; tight junctions, between L1–L2; L3–L4; L4-epithelial glial cell; and R7–R8. Septate junctions, between L1–L2, L3–L4, L3-, L4-, -, and an unidentified fibre making septate junctions with L1 and L2. Close appositions are found between R axons in the distal portion of the optic cartridges of this neuropil prior to extensive R chemical synapses with L1, L2. These loci (seen in freeze-fracture replicas) have rhomboidal patches of hexagonally arrayed P face particles.Intermembranous clefts between R axons are about 50 Å and are invariably electron lucent. These points of near contact between R terminals are probably the sites of low electrical resistance measured by Shaw (1979). Capitate projections are for the first time revealed in freeze fracture surfaces. Here epithelial glia send many, short, mushroom-shaped processes invaginating into R axons forming a tenacious structural bond. All four membrane leaflets (P and E faces of R axon and glial membrane) in the capitate projection possess particles in higher densities than in the surrounding nonspecialized regions. The known, general functions of each membrane specialization were correlated with the functional capacities of those lamina neurons possessing them in an effort to interpret better the integrative capacity of this neuropil. These data provide some fine structural bases for a putative blood-brain barrier between lamina and haemolymph, between lamina and peripheral retina, and possibly between lamina and second optic neuropil.     

Synapses between interneurons in the lateral geniculate nucleus of monkeys

Summary Electron microscopic examination of the monkey lateral geniculate nucleus, pars dorsalis (LGNd) reveals the occurrence of synaptic contacts between profiles belonging to interneurons (I-cells). Almost all combinations are observed, namely, axodendritic, dendrodendritic, dendrosomatic and somatodendritic, the most frequent being the dendrodentritic synapses between the presynaptic dendrites characteristic of I-cells. Quantitative analysis of 5 samples, each consisting of 200 m² of net LGNd neuropil, shows that presynaptic membrane specializations present in I-cell axonal and dendritic elements amount to a mean of 3.73 m or 8.16 % of the surface of such profiles. Only 61% of this extent is in contact with principal cells (P-cells), and an unexpectedly high 39% engages other I-cell elements. Approximately 18% of the neuropil is occupied by I-cell profiles. A tentative segregation of axonal and dendritic endings revealed the following significant mean differences: dendritic terminals are more numerous and larger; axonal profiles have more of their surface occupied by synaptic sites and each contact is longer. Findings suggest the existence of a network of interconnected interneurons which are presumably inhibitory in nature. Such an arrangement can provide a certain measure of anisotropic disinhibition which may be responsible for specific transformations occurring in the LGNd depending upon the size and velocity of the stimulus as well as of the degree of synchronicity of temporal patterns.     

Phenotypic Transitions of Macrophages Orchestrate Tissue Repair

Macrophages are essential for the efficient healing of numerous tissues, and they contribute to impaired healing and fibrosis. Tissue repair proceeds through overlapping phases of inflammation, proliferation, and remodeling, and macrophages are present throughout this progression. Macrophages exhibit transitions in phenotype and function as tissue repair progresses, although the precise factors regulating these transitions remain poorly defined. In efficiently healing injuries, macrophages present during a given stage of repair appear to orchestrate transition into the next phase and, in turn, can promote debridement of the injury site, cell proliferation and angiogenesis, collagen deposition, and matrix remodeling. However, dysregulated macrophage function can contribute to failure to heal or fibrosis in several pathological situations. This review will address current knowledge of the origins and functions of macrophages during the progression of tissue repair, with emphasis on skin and skeletal muscle. Dysregulation of macrophages in disease states and therapies targeting macrophage activation to promote tissue repair are also discussed.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Macrophages are essential to efficient healing of numerous tissues,^1–5 and they also contribute to impaired healing and fibrosis.^6–9 At different stages of the healing process, macrophages can promote debridement of the injury site, cell proliferation, angiogenesis, collagen deposition, and matrix remodeling, but improper regulation of any of these macrophage functions can also impair healing. These seemingly contradictory roles of macrophages are likely because of the ability of macrophages to assume a wide spectrum of functional phenotypes determined by their microenvironment and possibly by lineage, although the exact factors responsible for regulating macrophage phenotype in vivo remain poorly defined.^10–12Although macrophages in vivo rarely exhibit exactly the same phenotypes as cultured cells,^12,13 in vitro studies have defined convenient reference points along the nearly infinite spectrum of possible macrophage phenotypes.^10,14 Proinflammatory M1 macrophages are produced by exposure to interferon (IFN)γ and tumor necrosis factor (TNF)α or bacterial products, which activate MyD88 and NF-κB. M2a macrophages are produced by exposure to IL-4 or IL-13, which signal through the common receptor, IL-4 receptor α, and subsequent STAT6 activation; M2a macrophages have the potential to be profibrotic. Finally, immune regulatory functions have been postulated for macrophages of the M2b phenotype produced by combined stimulation with bacterial products and immune complexes, and for similar M2c phenotype produced by exposure to IL-10 or glucocorticoids, which act, in part, via inhibition of STAT1 and NF-κB.^10,14 During tissue repair, infiltrating macrophages do not appear to conform to in vitro–defined M1 and M2 categories^12,13,15; however, knowledge about the regulation and function of different macrophage phenotypes has the potential to yield new therapies for improving healing.This review will address the changing functions and phenotypes of macrophages during the progression of tissue repair, with emphasis on healing of skin and skeletal muscle. We also endeavor to point out gaps in the literature, particularly where macrophage function inferred from in vitro studies has not yet been confirmed in vivo. In addition, macrophage dysregulation during impaired healing will be discussed, as will potential therapies based on modulation of macrophage function.     

Trappin-2 Promotes Early Clearance of Pseudomonas aeruginosa through CD14-Dependent Macrophage Activation and Neutrophil Recruitment

Microaspiration of Pseudomonas aeruginosa contributes to the pathogenesis of nosocomial pneumonia. Trappin-2 is a host defense peptide that assists with the clearance of P. aeruginosa through undefined mechanisms. A model of macrophage interactions with replicating P. aeruginosa (strain PA01) in serum-free conditions was developed, and the influence of subantimicrobial concentrations of trappin-2 was subsequently studied. PA01 that was pre-incubated with trappin-2 (at concentrations that have no direct antimicrobial effects), but not control PA01, was cleared by alveolar and bone marrow-derived macrophages. However, trappin-2-enhanced clearance of PA01 was completely abrogated by CD14- null macrophages. Fluorescence microscopy demonstrated the presence of trappin-2 on the bacterial cell surface of trappin-2-treated PA01. In a murine model of early lung infection, trappin-2-treated PA01 was cleared more efficiently than control PA01 2 hours of intratracheal instillation. Furthermore, trappin-2-treated PA01 up-regulated the murine chemokine CXCL1/KC after 2 hours with a corresponding increase in neutrophil recruitment 1 hour later. These in vivo trappin-2-treated PA01 effects were absent in CD14-deficient mice. Trappin-2 appears to opsonize P. aeruginosa for more efficient, CD14-dependent clearance by macrophages and contributes to the induction of chemokines that promote neutrophil recruitment. Trappin-2 may therefore play an important role in innate recognition and clearance of pathogens during the very earliest stages of pulmonary infection.Nosocomial (hospital-acquired) infections are estimated to be responsible for up to 98,000 deaths per annum in the United States, with pneumonia the predominant cause of mortality.^1,2 The opportunistic pathogen Pseudomonas aeruginosa is the most commonly isolated microorganism from patients with nosocomial pneumonia in many published series.^3,4,5,6During hospital admission, the nasopharynx becomes colonized with potential pathogens such as P. aeruginosa. The likelihood of acquiring a potentially virulent colonizing organism increases with time.⁷ In turn, a significant proportion of nosocomial pneumonias are thought to arise from microaspiration of bacteria from the nasopharynx.⁸ Furthermore, aspiration of nasopharyngeal secretions in volumes likely to contain bacterial organisms takes place in normal patients during sleep.⁹ Therefore a greater understanding of the innate immune mechanisms operating against the very earliest invasion of the lung by small inocula of aspirated pathogens would aid the design of new preventive and therapeutic strategies for nosocomial pneumonia.Trappin-2 (also termed pre-elafin) is a 10-kDa cationic host defense peptide produced locally in the lung and in other mucosal sites exposed directly to the environment.^10,11 Trappin-2 has been shown to be directly anti-microbial against P. aeruginosa in vitro.^12,13,14 We previously showed that prophylactic overexpression of trappin-2 (which is absent in mice) enhances clearance of P. aeruginosa from murine lungs.¹⁵ However, the concentration of trappin-2 in bronchoalveolar lavage (BAL) fluid seemed to be significantly lower than that associated with direct antimicrobial activity.^12,13,14 These observations have prompted interest in other immunomodulatory activities for trappin-2.In this context, the immunomodulatory effects of trappin-2 may be divided into those involving innate immune priming and those that are anti-inflammatory. Thus trappin-2 can bind lipopolysaccharide (LPS), up-regulate LPS-induced tumor necrosis factor α (TNF-α) production from macrophages in vitro, and enhance LPS-induced leukocyte recruitment in vivo.^16,17 On the contrary, trappin-2 has been shown to attenuate nuclear factor kappa B (NF-κB) responses to oxidized low-density lipoprotein, LPS, and TNF-α in macrophages and endothelial cells.¹⁸ Recombinant C-terminal trappin-2 (also termed elafin) has been shown to prevent LPS-induced AP-1 and NF-κB activation through effects on the ubiquitin-proteasome pathway.¹⁹The early stages of acute P. aeruginosa lung infection are thought mainly to involve interactions between the alveolar macrophage (AM, the patrolling phagocytic cell of the alveolar space) and the microbe. These interactions are thought to be independent of serum opsonins, given the low levels of such opsonins in the lung before injury. Two distinct receptors, complement receptor 3 (CR-3) and CD14, have been shown to mediate nonopsonic phagocytosis of different P. aeruginosa strains.²⁰ Less is known about the ligands that interact with these receptors in the lung during the initial stages of infection when serum-derived ligands in the alveolar space are likely to be present at ineffective concentrations.We hypothesized that trappin-2 may play a role in the early clearance of P. aeruginosa through an influence on macrophages. Using in vitro and in vivo assays we studied P. aeruginosa-macrophage interaction at very early stages of lung infection when bacteria are present at low numbers and the effect of serum and mucosal antibodies are considered to be minimal. Our studies demonstrate for the first time that trappin-2 at subantibacterial concentrations can increase suppression of P. aeruginosa growth by macrophages. Our studies in AMs (which are inherently CR-3-deficient) and macrophages genetically engineered to be CD14-deficient suggest that trappin-2-treated P. aeruginosa binds to membrane CD14. Infection of mice with low doses of trappin-2-treated P. aeruginosa [∼10⁵ colony forming units (CFU)] showed that this effect is maintained in vivo and that trappin-2 amplifies host defense by the specific recruitment of neutrophils into the lung during early infection.     

Ultrastructure of a large ventro-lateral dendritic bundle in the rat ventral horn

Summary There is an extensive bundle of dendrites with a rostro-caudal axis in the ventro-lateral lamina of the sixth lumbar segment in each side of the rat spinal cord. Such a bundle has a diameter of about 250 m and contains over 1400 parallel dendrites., each with a diameter of less than 8 m, interspersed between neuronal somata. The volume fraction of dendrites in the bundle neuropil is about 55%, the remainder being equally distributed between astrocytes, synaptic boutons, and axons, most of which are unmyelinated. An analysis of the median percentage covering of dendrites by contiguous elements of the neuropil reveals that as the dendrite diameter decreases from 4 to 0.2 m (mean=2 m), astrocytes increase from 43 to 75%, axons decrease from 21% to zero, boutons decrease from 28% to zero, and dendrites decrease from 10% to zero. There is a mean of 18 synaptic boutons per 100 n² of the overall dendritic surface, but larger boutons tend to be more frequent on larger dendritic profiles. Apposed dendrites and their somata may have either puncta adhaerentia or confronting subsurface cisternae. Synaptic types in the rat are similar to those reported for the cat. The morphological findings are discussed with respect to previously proposed interaction between neural elements.     

Alternatively Activated Macrophages and Collagen Remodeling Characterize the Postpartum Involuting Mammary Gland across Species

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non–pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.Although it is recognized that full-term pregnancy at an early age reduces the lifetime risk of developing breast cancer, women of all ages have a transient increase in breast cancer risk with a recent pregnancy.^1–4 Breast cancers diagnosed up to five years out from a completed pregnancy have been referred to as pregnancy-associated or PABC.^5,6 Several studies have shown that PABC frequently metastasizes, resulting in poor prognosis for the patient.^6–8 Epidemiological data identify women whose breast cancer is diagnosed postpartum, rather than during pregnancy, as having the worst outcomes.^7–14 Further, when breast cancer patients were matched for known prognostic indicators, the postpartum window proved to be an independent factor for metastasis, whereas a diagnosis during pregnancy did not.^11,14,15 We have proposed the involution-hypothesis to account for the high metastatic occurrence of PABC diagnosed during the postpartum window.^5,16–18 Specifically, we predict that the involuting microenvironment, with its similarities to wound and cancer environments, supports dissemination of pre-existing but previously quiescent tumor cells. In support of this hypothesis, we have shown that extracellular matrix (ECM) proteins isolated from the involution microenvironment have wound healing characteristics and are promotional for breast tumor cell metastasis.¹⁶ We extend our studies to address whether alternatively activated M2 macrophages, an immune cell type implicated in wound healing and breast cancer metastasis, participate in postpartum gland regression.Postpartum mammary gland involution is a necessary physiological process required to return the lactation competent gland to a nonlactating state poised to respond to a subsequent round of pregnancy. In rodent models, weaning-induced milk stasis triggers cessation of milk secretion and the milk-producing secretory alveoli that developed during pregnancy are rapidly resorbed. In rodent models, it has been shown that 50% to 80% of the secretory mammary epithelium is eliminated by apoptosis within the first week of involution.^19,20 This dramatic tissue remodeling requires two major processes: apoptotic death of the mammary epithelial cells and stromal activation.²¹ Accumulating evidence in rodents indicates that tissue-remodeling programs similar to wound healing are used to remodel the lactating gland to its postpartum state. Similarities include elevated levels of the immunomodulators transforming growth factor (TGF)-β1 and 3, increases in matrix metalloproteinase (MMP)-2, −3, and −9, deposition of fibrillar collagens, presence of bioactive proteolytic fragments of the ECM proteins fibronectin and laminin, as well as gene expression profiles consistent with activation of innate and adaptive immunity.^22–25Macrophages are a dynamic population of immune cells recognized for their contribution to wound resolution through phagocytosis and for their role in antigen presentation. The detailed functions of macrophages are extensive, as their precursor cells can respond to a variety of physiological situations and mature along a spectrum of phenotypes. The M1/M2 macrophage nomenclature system described by Mantovani and colleagues²⁶ primarily divides cells based on production of Th1 versus Th2 cytokines. According to this classification, the M1-type, or classically activated macrophages, mature in response to signaling molecules elicited by intracellular pathogens including lipopolysaccharide, interferon-γ and -β, and TNF-α. These M1 macrophages respond in kind by producing Th1 cytokines including interleukin (IL)-12 and IL-23 that subsequently promote activation of cytotoxic T-lymphocytes. M1 macrophages are involved in antigen presentation, immune surveillance, and killing of cells with foreign antigens, including tumor cells.^26–28On the other end of the spectrum, M2 macrophages are alternatively activated by Th2 cytokines such as IL-4, IL-13, and the immunosuppressive IL-10.²⁶ M2-type macrophages in turn produce Th2 cytokines. M2 macrophages participate in wound healing and tissue repair through activities including phagocytic debris clearance, release of TGF-β and fibronectin, and release of PDGF and VEGF,^27,29 which promote angiogenesis. Recently, it has become apparent that macrophages are functionally plastic, in that they respond to their environment with heterogeneous and transient phenotypes that are not distinctly divided into the M1/M2 categories.³⁰Several recent reviews highlight shared characteristics of M2 macrophages and tumor-associated macrophages (TAMs).^26,31 TAMs are important in the progression of mammary cancer in several rodent models.^32,33 The observed similarities between TAMs and M2 macrophages suggest that M2 macrophages may also have tumor promotional capabilities.³⁴ Further, clinical data reveals that macrophage chemotactic factors, macrophage growth factors, and macrophage infiltration all correlate with negative outcomes for breast cancer patients.^35–40 TAMs and M2 macrophages are thus implicated as key mediators of breast cancer progression.Based on evidence that macrophages promote breast cancer and that the wound-healing microenvironment of the involuting rodent mammary gland is tumor promotional, we evaluated whether macrophages characterize the postpartum involuting mammary gland. Here we demonstrate an influx of macrophages during postlactational involution, identified as consisting primarily of M2 macrophages in rodents and humans. Further, we provide a plausible mechanism for macrophage recruitment via their attraction to the ECM protein collagen I. These observations are consistent with our previous data demonstrating that the involuting rat mammary gland is characterized by a wound healing microenvironment and extends these findings to human breast tissues. These data further support the hypothesis that postpartum involution promotes tumor progression and accounts for the poor prognosis of PABC.     

Macrophages Protect against Muscle Atrophy and Promote Muscle Recovery in Vivo and in Vitro : A Mechanism Partly Dependent on the Insulin-Like Growth Factor-1 Signaling Molecule

Hindlimb unloading and reloading are characterized by a major loss of muscle force and are associated with classic leukocyte infiltration during recovery from muscle atrophy. Macrophages act as a cellular cornerstone by playing both pro- and anti-inflammatory roles during muscle recovery from atrophy. In the present study, we investigated the role of macrophages in muscle atrophy and regrowth using in vivo and in vitro models. Mice depleted in monocytes/macrophages and submitted to a hindlimb unloading and reloading protocol experienced a significant delay in muscle force recovery compared with matched placebo mice at 7 and 14 days after reloading. Furthermore, an in vitro myotube/macrophage coculture showed that anti-inflammatory macrophages, which contain apoptotic neutrophils and express low levels of cyclooxygenase-2, completely prevented the loss of protein content and the myotube atrophy observed after 2 days in low serum medium. The presence of macrophages also protected against the decrease in myosin heavy chain content in myotubes exposed to low serum medium for 1 day. Interestingly, the addition of an anti-IGF-1 antibody to the coculture significantly decreased the ability of macrophages to protect against myotube atrophy and myosin heavy chain loss after 2 days in low serum medium. These results clearly indicate that macrophages and, more precisely, the release of IGF-1 by macrophages, play an important role in recovery from muscle atrophy.Monocytes are large mononuclear cells that circulate in the blood and differentiate into macrophages in invaded tissues in response to various stimuli.¹ Macrophages have a strong phagocytic capacity and can orchestrate the inflammatory process via the release of a wide variety of cytokines and chemokines such as interleukin (IL)-1, tumor necrosis factor-α, and macrophage inflammatory protein-2.^2,3 Numerous studies have also demonstrated that macrophages play a direct role in tissue recovery through the release of the anti-inflammatory molecules and anabolic growth factors IL-10, basic fibroblast growth factor, and insulin-like growth factor-1 (IGF-1).^4,5,6The regulation of the multiple and occasionally opposing functions of macrophages is very complex and poorly understood.^7,8,9 To add to this complexity, the diversity of experimental models can also lead to different conclusions regarding the roles of each macrophage phenotype in muscle recovery from atrophy or damage. For example, in a model of eccentric contractions, in which injured muscle is basically devoid of neutrophils, macrophage invasion contributes to secondary damage to the skeletal muscle.¹⁰ On the other hand, the phagocytosis of apoptotic neutrophils and necrotic cells by macrophages induces a change in macrophage phenotype from pro- to anti-inflammatory, which has a strong modulatory effect on cytokine profiles and is essential for dampening environmental inflammatory signals.^11,12 Regarding the different subsets of macrophages, coculture experiments have shown that pro-inflammatory macrophages (Ly-6C^hi) stimulate myogenic cell proliferation, whereas anti-inflammatory macrophages (Ly-6C^lo) exert a strong differentiating influence on myogenic cells.^12,13 Nonetheless, in a model in which rodent hindlimbs are deprived of mechanical loading for 10 days followed by reloading, the high concentration of leukocytes in reloaded muscles regardless of the absence of significant muscle damage raises intriguing questions about the detrimental and beneficial roles of leukocytes in muscle dysfunction and recovery from atrophy.^14,15,16 It is thus tempting to speculate that the roles of macrophages may vary as a function of the type of insult.In the present study, mice depleted in macrophages were submitted to hindlimb unloading and reloading to evaluate the roles of macrophages in muscle atrophy and regrowth. Our results showed that macrophages neither prevent the loss in muscle force nor promote recovery during the early inflammatory phase (1 and 3 days after reloading). However, they play a key role in muscle growth and recovery at later times (7 and 14 days after reloading). In addition, an in vitro coculture model in which atrophied myotubes were combined with macrophages expressing an anti-inflammatory phenotype showed that the presence of macrophages protects myotubes from atrophy and that this protective effect is partly mediated by the release of IGF-1.     

Macrophages Are Essential for the Early Wound Healing Response and the Formation of a Fibrovascular Scar

After wounding, multiple cell types interact to form a fibrovascular scar; the formation and cellular origins of these scars are incompletely understood. We used a laser-injury wound model of choroidal neovascularization in the eye to determine the spatiotemporal cellular events that lead to formation of a fibrovascular scar. After laser injury, F4/80⁺ myeloid cells infiltrate the wound site and induce smooth muscle actin (SMA) expression in adjacent retinal pigment epithelial cells, with subsequent formation of a SMA⁺NG2⁺ myofibroblastic scaffold, into which endothelial cells then infiltrate to form a fibrovascular lesion. Cells of the fibrovascular scaffold express the proangiogenic factor IL-1β strongly, whereas retinal pigment epithelial cells are the main source of VEGF-A. Subsequent choroidal neovascularization is limited to the area demarcated by this myofibroblastic scaffold and occurs independently of epithelial- or myeloid-derived VEGF-A. The SMA⁺NG2⁺ myofibroblastic cells, F4/80⁺ macrophages, and adjacent epithelial cells actively proliferate in the early phase of the wound healing response. Cell-lineage tracing experiments suggest that the SMA⁺NG2⁺ myofibroblastic scaffold originates from choroidal pericyte-like cells. Targeted ablation of macrophages inhibits the formation of this fibrovascular scaffold, and expression analysis reveals that these macrophages are Arg1⁺YM1⁺F4/80⁺ alternatively activated M2-like macrophages, which do not require IL-4/STAT6 or IL-10 signaling for their activation. Thus, macrophages are essential for the early wound healing response and the formation of a fibrovascular scar.The acute wound healing response after injury is a complex process involving multiple cell types that interact in a highly coordinated fashion to form a fibrovascular scar. The cell types and the spatiotemporal events involved in the process of forming a fibrovascular scar are tissue-specific and are not well understood. We used an acute wound healing model of choroidal neovascularization (CNV) in the eye to determine the spatiotemporal cellular events that lead to the formation of a fibrovascular scar and used cell-lineage tracing and various genetic mouse models to determine cell type-specific functions in this process. This mouse wound healing model is a frequently used experimental model for CNV, the cardinal feature of neovascular (often called wet) age-related macular degeneration (AMD), and involves a laser-induced acute wounding of the retinal pigment epithelium (RPE)–choroid interface with disruption of the RPE focally.¹ In this experimental model, a myeloid cell infiltrate occurs early after laser injury and is followed by the formation of neovessels, which originate mainly from the choroidal vasculature.² It has been proposed that macrophages affect this neovascularization process. Clodronate liposome–induced ablation of macrophages inhibited CNV lesion formation²; however, others have suggested that macrophages promote CNV, and thus the role of macrophages in this model is not well understood.³ Although some studies suggest that macrophages can undergo transdifferentiation into endothelial cells during induced angiogenesis, it is not known whether macrophages that accumulate locally at the site of laser injury undergo endothelial transdifferentiation and incorporate into neovessels in CNV.^4,5 Whether macrophages undergo activation in CNV lesions and polarize to either an M1- or M2-type macrophage population also remains to be determined; however, in tumor angiogenesis, a proangiogenic role of alternatively activated M2-type macrophages that express multiple proangiogenic growth factors and cytokines has been proposed.⁶ Similarly, it has been suggested that the proangiogenic growth factor VEGF-A is critical for CNV formation and that infiltrating macrophages express VEGF-A and promote angiogenesis in neovascular AMD,⁷ but others have found VEGF-A expression only in RPE cells in neovascular AMD lesions, but not in macrophages.⁸ Thus, whether macrophages promote CNV lesions through VEGF-A expression is not known and has not been shown in this laser-injury model.Importantly, although this experimental laser-injury CNV model is frequently used and described as a model for neovascular AMD, the acute laser injury occurs in the setting of healthy RPE cells, in contrast to the chronic disease process in AMD. Thus, an understanding of the spatiotemporal cellular events that occur after laser injury at the RPE–choroid interface is important, both to allow drawing comparisons between this acute laser-injury model and the chronic pathology of neovascular AMD and to gain insights into the role of various cell populations during the early wound healing response. Here, we characterize the spatiotemporal cellular events in laser injury-induced CNV and provide evidence for a critical role of macrophages in the early wound healing response and fibrovascular scar formation.     

Macrophage Matrix Metalloproteinase-9 Mediates Epithelial-Mesenchymal Transition in Vitro in Murine Renal Tubular Cells

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial−mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-β. Transforming growth factor-β-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.Interstitial macrophage infiltration is a hallmark of all progressive renal diseases regardless of the initial cause of the injury.^1,2 Macrophages have long been known to play an important role in renal fibrosis,³ which is a central component of the final common pathway leading to renal failure. Previous studies have demonstrated a close association between macrophage infiltrate and excessive extracellular matrix protein accumulation in diseased human kidney as well as in experimental models.^4–6 In addition, the number of infiltrating macrophages has been shown to correlate well with the number of myofibroblasts,^7,8 the effector cells responsible for secretion of extracellular matrix proteins. A recent study revealed that blockade of macrophage recruitment in obstructive renal injury resulted in a reduction in renal fibrosis via tubular cell epithelial−mesenchymal transition (EMT),⁹ which has been recognized as an important source of myofibroblasts in renal fibrosis. However, the exact mechanism underlying the contribution of macrophages to renal fibrosis via tubular cell EMT remains undefined. As a major source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages may be major determinants of the outcome of renal fibrosis.Tubular cell EMT is a process by which tubular epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype. This process has been recognized as one of several pathways contributing to the myofibroblast population in renal fibrosis.¹⁰ Despite emerging and conflicting evidence about the relative importance of various sources of myofibroblasts,^11,12 it is generally accepted that tubular cell EMT plays an important role in renal fibrosis. Since the concept of tubular cell EMT was first proposed, numerous studies have provided evidence for tubular cell EMT in various experimental models as well as in human biopsies.¹⁰ Furthermore, the importance of tubular cell EMT has been demonstrated by Iwano et al¹³ using transgenic mice and direct genetic tagging of tubular epithelial cells to show that more than a third of myofibroblasts in kidneys with unilateral ureteral obstruction are derived from tubular epithelial cells via tubular cell EMT. Moreover, blockade of tubular EMT has been shown to attenuate renal fibrosis in obstructive nephropathy.¹⁴ However, some controversy remains as to whether tubular cell EMT plays a consistent role in other experimental models, and its exact contribution in renal fibrosis is yet to be established.Although pro-fibrogenic growth factors are well known as inducers of tubular cell EMT, cumulative evidence suggests an important role for MMPs. Traditionally, MMPs were thought to be antifibrogenic due to their ability to degrade extracellular matrix proteins, yet MMPs—in particular MMP-2 and MMP-9—have been recognized as promoters of tubular cell EMT via basement membrane disruption. In fact, induction of tubular cell EMT in vitro¹⁵ and in vivo¹⁴ has been shown to be associated with increased expression of MMP-2 and MMP-9. Earlier studies have demonstrated that tubular epithelial cells undergoing mesenchymal transition are closely associated with damaged tubular basement membrane and that complete transition requires tubular basement membrane damage.¹⁶ Later studies have shown directly that MMPs can disrupt basement membrane integrity; loss of MMP-9 expression lead to preservation of basement membrane integrity and inhibition of tubular cell EMT in obstructed kidney of tissue type plasminogen activator knockout mice.¹⁴ Despite this evidence supporting induction of tubular cell EMT by MMPs, the precise contribution of MMPs may have been underestimated. In cancer research, MMPs are well known to directly induce EMT in tumor cells of epithelial origin and to promote tumor progression via basement membrane disruption.¹⁷ MMP-2 has been shown consistently to be necessary and sufficient to induce tubular cell EMT in a rat tubular epithelial cell line (NRK52e).¹⁸ In addition, recent studies from our laboratory have demonstrated that MMP-3 and MMP-9 are also capable of inducing tubular cell EMT in NRK52e cells via the disruption of the cell adhesion molecule E-cadherin. Finally, the fact that transforming growth factor (TGF)-β-induced tubular cell EMT in NRK52e was inhibited by a broad spectrum MMP inhibitor suggests a primary role of MMP in TGF-β-induced tubular cell EMT.¹⁹ Together, these data suggest that MMPs from macrophages may play a major role in induction of tubular cell EMT. Therefore the aim of this study was to investigate the contribution of macrophages and their secreted MMPs to the induction of tubular cell EMT.     

Ecto-ATPase activity on the surface of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and its possible role in the parasite–host cell interaction

This study describes the possible role of Mg²⁺-dependent ecto-ATPase activity on the Trypanosoma cruzi–host cell interaction. Mg²⁺-dependent ecto-ATPase activity is observed on the cell body and flagellar membranes of the parasite and is about 20 times greater in trypomastigotes, as compared with epimastigotes. Suramin (a competitive antagonist of P2 receptors) and the impermeant agent 4,4-diisothiocyanostylbene 2,2-disulfonic acid (DIDS), both inhibitors of ecto-ATPases, strongly inhibited ATPase activity and the adhesion and internalization of both evolutive forms by mouse resident macrophages. Suramin inhibited the growth of epimastigotes, suggesting a direct participation of ecto-ATPase activity in this process. To overcome the presence of suramin in the culture medium during the time of growth, Mg²⁺ ecto-ATPase activity was enhanced 4-fold, as compared with control parasites. The over-expression in enzyme activity was followed by a dramatic increase in the adhesion of epimastigotes to resident macrophages above the level observed for non-treated parasites.     

Differential expression of RARbeta isoforms in the mouse striatum during development: a gradient of RARbeta2 expression along the rostrocaudal axis.

The retinoic acid receptor RARbeta is highly expressed in the striatum of the ventral telencephalon. We studied the expression pattern of different RARbeta isoforms in the developing mouse striatum by in situ hybridization. We found a differential ontogeny of RARbeta2 and RARbeta1/3 in embryonic day (E) 13.5 lateral ganglionic eminence (striatal primordium). RARbeta2 mRNA was detected primarily in the rostral and ventromedial domains, whereas RARbeta1/3 mRNAs were enriched in the caudal and dorsolateral domains. Notably, by E16.5, a prominent decreasing gradient of RARbeta2 mRNA was present in the developing striatum along the rostrocaudal axis, i.e., RARbeta2 was expressed at higher levels in the rostral than the caudal striatum. No such gradient was found for RARbeta1/3 and RARbeta3 mRNAs. The rostrocaudal RARbeta2 gradient gradually disappeared postnatally and was absent in the adult striatum. The differential expression pattern of RARbeta isoforms in the developing striatum may provide an anatomical basis for differential gene regulation by RARbeta signaling.     

The corticothalamic projection from the gyrus proreus and the medial wall of the rostral hemisphere in the cat an experimental study with silver impregnation methods

Summary The corticothalamic projections from the gyrus proreus and the medial wall of the rostral hemisphere have been studied in the cat with the silver method of Nauta. The gyrus proreus projects upon the following nuclei (for abbreviations, see list on page 133), ipsilateral R, VA, VM, VL, MD, Pc, CL, CM, Pf, VPM, VPMpc. VPI and to the contralateral principal nucleus of the trigeminal nerve. The medial wall of the rostral hemisphere projects bilaterally upon R, VA, VM, VL, MD, Pc, CL, CM, Pf, VPM, VPMpc, VPI, VPL, the dorsal column nuclei and the principal nucleus of the trigeminal nerve. The ipsilateral thalamic projection is more abundant than the contralateral. The latter appears to increase in amount as the lesion is placed successively more ventrally on the medial wall of the rostral hemisphere. Some degenerating fibers cross in the corpus callosum and descend in the contralateral internal capsule but the majority cross in the dorsal part of the anterior commissure and reach the medial aspect of the anterior limb of the contralateral internal capsule. A somatotopical organization of the medial wall of the rostral hemisphere has been demonstrated. The rostrocaudal part projects upon the ipsilateral VPL lateralis (VPLl) and nucleus cuneatus and the contralateral nucleus gracilis and VPL medialis (VPLm). The caudal part of this cortical area sends fibers bilaterally to VPM, VPMpc, and the principal nucleus of the trigeminal nerve. The intermediate part, which also includes agranular cortex on the medial wall, projects upon ispsilateral VPLm and nucleus gracilis and upon contralateral VPLl and nucleus cuneatus. — The fibers to the ventro-basal complex, dorsal column nuclei and the principal nucleus of the trigeminal nerve are rather thick. The corticofugal fibers to the other thalamic nuclei are quite thin. — The findings are discussed in light of relevant anatomical and physiological observations in the literature and special emphasis has been laid on reported observations on the supplementary motor area.     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

Involvement of rostral ventromedullar neuronal structures in nitric oxide modulation of central chemosensitive drive

Surface perfusion of the rostral ventromedullar cerebral subdivisions with artificial cerebrospinal fluid containing exogenous NO donor sodium nitroprusside (0.1 mM) increased the discharge rate of the phrenic nerve and potentiated the response of the respiratory center to hypercapnia in narcotized mature rats. The latter reaction was prevented by blockage of NO-synthase in rostral ventromedullar neural structures with N-nitro-L-arginine methyl ester (L-NAME, 0.3 mM). It was hypothesized that rostral ventromedullar neural structures are involved in modulatory action of NO on central chemosensitive drive.