Cryopreservable neutrophil surrogates: granule-poor, motile cytoplasts from polymorphonuclear leukocytes home to inflammatory lesions in vivo

Cytokineplasts (CKP) are anucleate, motile, granule-poor fragments induced from polymorphonuclear leukocytes on surfaces by the brief application of heat. Derived from the peripheral cytoplasm and membranes of PMN, they retain the sensing, transducing, and effector mechanisms necessary for chemotaxis and phagocytosis, and appear to represent a functional, self-purification of the motile apparatus. Unlike their parent PMN, CKP are cryopreservable. We have shown that they can adhere to endothelial cell monolayers, open interendothelial cell junctions, and migrate to the abluminal side when stimulated by a chemoattractant. Employing an animal model, we now show that, given intravenously, they can home to an inflammatory target lesion in vivo.     

Functional integrity of cytokineplasts: specific chemotactic and capping responses

Cytokineplasts (CKP) are motile, membrane-bound, anucleate, granule-poor cytoplasmic fragments that are induced from human blood polymorphonuclear leukocytes (PMN) by the brief application of heat. We examined CKP with respect to specific chemotactic and capping responses, the presence of the N-formyl-peptide chemotactide receptor, and evidence of respiratory burst activity and compared them with CB-cytoplasts, which are fragments created by the centrifugation of cytochalasin B (CB)-treated PMN at high speeds. Under agarose, CKP responded chemotactically to both N-formyl-methionyl-leucyl-phenylalanine (fmlp) and zymosan-activated serum; CB-cytoplasts responded to neither chemoattractant. Despite the functional differences, both fragments retained N-formyl-peptide receptors as measured by affinity labeling with N-formyl-norleu-leu-phe-norleu-125I-tyr-lys and autoradiography of dried SDS-PAGE gels. For studies of capping we used a murine monoclonal antibody, PMN7C3, which binds a specific, widely distributed membrane component of intact PMN, and on warming, promptly induces capping of ligand-receptor complexes. Rhodamine-conjugated PMN7C3 at 4 degrees C labeled the surface of CKP homogeneously. As the CKP warmed to 37 degrees C, label became concentrated in small fluorescent caps at the rear of migrating fragments. Although CB-cytoplasts also bound the fluorochromed antibody homogeneously in the cold, on warming they were unable to concentrate the label normally. With respect to respiratory burst activity, the situation in the two fragments was reversed: CKP did not generate superoxide anion when stimulated either with phorbol myristate acetate or with fmlp after pretreatment with CB; CB-cytoplasts, as noted earlier by other investigators, did. These two types of cytoplasts with markedly different capabilities have complementary roles in the analysis of PMN function.     

Clonogenic assay of type a influenza viruses reveals noninfectious cell-killing (apoptosis-inducing) particles

Clonogenic (single-cell plating) assays were used to define and quantify subpopulations of two genetically closely related variants of influenza virus A/TK/OR/71 that differed primarily in the size of the NS1 gene product; they expressed a full-size (amino acids [aa] 1 to 230) or truncated (aa 1 to 124) NS1 protein. Monolayers of Vero cells were infected with different amounts of virus, monodispersed, and plated. Cell survival curves were generated from the fraction of cells that produced visible colonies as a function of virus multiplicity. The exponential loss of colony-forming capacity at low multiplicities demonstrated that a single virus particle sufficed to kill a cell. The ratios of cell-killing particles (CKP) to plaque-forming particles (PFP) were 1:1 and 7:1 in populations of variants NS1(1-124) and NS1(1-230), respectively. This study revealed a new class of particles in influenza virus populations-noninfectious CKP. Both infectious and noninfectious CKP were 6.3 times more resistant to UV radiation than PFP activity. Based on UV target theory, a functional polymerase subunit was implicated in a rate-limiting step in cell killing. Since influenza viruses kill cells by apoptosis (programmed cell death), CKP are functionally apoptosis-inducing particles. Noninfectious CKP are present in excess of PFP in virus populations with full-size NS1 and induce apoptosis that is temporally delayed and morphologically different than that initiated by infectious CKP present in the virus population expressing truncated NS1. The identification and quantification of both infectious and noninfectious CKP defines new phenotypes in influenza virus populations and presents a challenge to determine their role in regulating infectivity, pathogenesis, and vaccine efficacy.     

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by > or =10(4) bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS > intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.     

Isolation of the saprophytic strain of MC-3 and participation of the cell surface structure in predation

From a predatory bacterium, MC-3, a mutant strain which lost predation ability was isolated by chance selection. Biological properties of the mutant were the same as the parent except only saprophytic property. Properties of the parent and the mutant strains of MC-3, such as bacteriolytic activity of the culture supernatant, digestion of peptidoglycan of the host bacteria, and growth by utilizing the host cells or their cytoplasmic substances, suggested that cell surface structure of the host cell plays an important role in predation and host specificity.     

Interleukin 1 production by human polymorphonuclear neutrophils

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.     

Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent

The recruitment of polymorphonuclear leukocytes (PMN) from the vascular space to the alveolar air space is an early event in host defense against pneumococcal pneumonia. Pneumolysin is a virulence factor for Streptococcus pneumoniae, but a specific role for pneumolysin in neutrophil-endothelial cell interactions has not been investigated. Using a Transwell system, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to wild-type S. pneumoniae (D39) and a pneumolysin-deficient mutant (plnA(-)) incubated on the abluminal surface of the monolayer. S. pneumoniae induction of PMN migration was dose dependent and elicited by > or =10(5) D39. Mutants lacking pneumolysin had dramatically reduced potency for eliciting PMN migration compared with the parent strain (5 x 10(6) plnA(-) elicits 18.6% PMN migration vs. 55.5% for 5 x 10(6) D39). The disparity between D39 and plnA(-) persisted in ethanol-fixed bacteria, consistent with the properties of pneumolysin. Neither conditioned medium from D39 nor purified pneumolysin elicited PMN migration to the same extent as the intact D39, suggesting that the role of pneumolysin in eliciting PMN migration requires a more complex interaction between the organism, the endothelium, and the PMN. Both D39 and plnA(-) adhered to, and translocated across, the endothelium in the abluminal to luminal direction and elicited similar levels of IL-8 production. Neither strain elicited upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin, and they did not cause translocation of NF-kappaB to the nucleus. These findings demonstrate a novel role for pneumolysin in pneumococcus-induced PMN recruitment across the pulmonary endothelium.     

Plunder of Human Blood Leukocytes Containing Ingested Material,by Other Leukocytes: Where Is the Fusagen That Allows Preservation of Membrane Integrity and Motile Function?

In studying phagocytosis of zymosan particles by human blood monocytes in phase-contrast videomicroscopy, we found that monocytes loaded with zymosan particles became chemotactic for polymorphonuclear leukocytes (PMN) which closed on them and purloined their particle content. This despoliation usually occurred in monocytes that had begun to swell—prefiguring their death. The violent seizure of their contents by the aggressing PMN often tore the monocytes apart. However, some apparently healthy monocyte survived the removal of zymosan content by PMN or, more commonly, its removal by another monocyte. PMN—a much hardier cell in slide preparations—that were similarly loaded with zymosan particles, also attracted PMN. The latter could remove zymosan from the target cell without killing it. Thus, leukocytes were sacrificing significant portions of themselves without losing residual membrane integrity and motile function. Their behavior with respect to other particles (e.g., bacteria) will be of interest. We suggest that the membrane fusagen resides in the inner membrane leaflets when they are brought together in an extreme hourglass configuration. This event may be similar to the fragmentation of erythrocytes into intact pieces, the formation of cytokineplasts, the rear extrusion of content by migrating cells on surfaces, and the phagocytic process itself.     

SIRPbeta1 is expressed as a disulfide-linked homodimer in leukocytes and positively regulates neutrophil transepithelial migration

Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (beta) or inhibitory (alpha) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRPalpha binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRPalpha and SIRPbeta isoforms expressed in PMN include Bit/PTPNS-1 and SIRPbeta1, respectively. Furthermore, although SIRPalpha (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRPbeta1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRPbeta1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRPalpha (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRPbeta1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10-12-kDa protein associated with SIRPbeta1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor gamma chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Efficient phagocytosis of Klebsiella pneumoniae strains that poorly bind to human polymorphonuclear leukocytes.

The phagocytosis process of unencapsulated MIAT-negative strains that, although binding very poorly to human polymorphonuclear leukocytes (PMN) at 4 degrees C, are efficiently killed by these cells at 37 degrees C, was studied. At 37 degrees C the number of bacteria bound to the PMN external surface was similar to that observed at 4 degrees C (about 100 bacteria/100 PMN after 60 min); on the contrary the number of internalized bacteria was much higher (from 500 bacteria/100 PMN after 60 min). Interactions between phagocytosis-sensitive Klebsiella pneumoniae strains (PSK) and PMN were then compared with those of two isogenic Escherichia coli strains with and without type 1 fimbriae. Whereas PSK strain binding to blocked PMN was very slow and became significant only after 5-6 h, that of phagocytosis-sensitive fimbriated E. coli was rapid and efficient. Phagocytosis-resistant, non fimbriated E. coli strain bound with an efficiency that, within the first 60 min, was not very different from that of the PSK strains. However, longer incubations led to increases in PSK binding, whereas unfimbriated E. coli remained constant. PSK and fimbriated E. coli strains were efficiently internalized and killed, whereas the unfimbriated E. coli strain was not. It is suggested that PMN can phagocytize unopsonized bacteria through two different mechanisms. By one mechanism, observed with the fimbriated E. coli strain, PMN bind many more bacteria than those they can internalize. By the other, observed with PSK strains, PMN bind only the bacteria they can immediately internalize.     

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Abstract Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in FcγIIR, FcγIIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CD11b (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

Incompetence of neutrophils to invasive group A streptococcus is attributed to induction of plural virulence factors by dysfunction of a regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection.     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

Bacteria Induce Prolonged PMN Survival via a Phosphatidylcholine-Specific Phospholipase C- and Protein Kinase C-Dependent Mechanism

Polymorphonuclear leukocytes (PMNs) are essential for the human innate immune defense, limiting expansion of invading microorganisms. PMN turnover is controlled by apoptosis, but the regulating signaling pathways remain elusive, largely due to inherent differences between mice and humans that undermine use of mouse models for understanding human PMN biology. Here, we aim to elucidate signal transduction mediating survival of human peripheral blood PMNs in response to bacteria, such as Yersinia pseudotuberculosis, an enteropathogen that causes the gastro-intestinal disease yersiniosis, as well as Escherichia coli and Staphylococcus aureus. Determinations of cell death reveal that uninfected control cells undergo apoptosis, while PMNs infected with either Gram-positive or -negative bacteria show profoundly increased survival. Infected cells exhibit decreased caspase 3 and 8 activities, increased mitochondrial integrity and are resistant to apoptosis induced by a death receptor ligand. This bacteria-induced response is accompanied by pro-inflammatory cytokine production including interleukin-8 and tumor necrosis factor-α competent to attract additional PMNs. Using agonists and pharmacological inhibitors, we show participation of Toll-like receptor 2 and 4, and interestingly, that protein kinase C (PKC) and phosphatidylcholine-specific phospholipase C (PC-PLC), but not tyrosine kinases or phosphatidylinositol-specific phospholipase C (PI-PLC) are key players in this dual PMN response. Our findings indicate the importance of prolonged PMN survival in response to bacteria, where general signaling pathways ensure complete exploitation of PMN anti-microbial capacity.     

Chemical, Biological, and Structural Properties of Stable Proteus L Forms and Their Parent Bacteria

Proteus L forms were disrupted by osmotic shock, and the sedimentable material present in the homogenate was further fragmented in a Sorvall pressure cell. The pressure cell was also used for disrupting normal Proteus cells. The homogenates obtained were fractionated by differential centrifugation. Purified endotoxins were isolated from the major fractions by phenol extraction. Material extracted with phenol from the membrane fraction of the L forms was about as toxic and pyrogenic on a weight basis as the typical enterobacterial endotoxins isolated from cell walls of normal bacteria. The yield of extract from L forms was about one-third of that from an equal weight of normal bacteria. No differences in the gross chemical composition of the phenol extracts from the L forms and the normal cells could be ascertained. A close serological relationship existed between extracts obtained from two L forms and their respective parent bacteria, but no such relationship was found in the case of the third L form studied and its parent bacterium. Diaminopimelic acid was not detected in the membranes of the L forms, but these membranes contained most of the succinic dehydrogenase of the organisms. Only small amounts of this enzyme were present in the wall fraction of normal bacteria. The data obtained suggest that precursors of the Proteus endotoxins are formed either in the soluble protoplasm of normal cells and L forms or at sites on the membrane from which they are readily liberated into the protoplasm, whereas the final steps of the synthesis of these toxins take place at the cytoplasmic membrane. In normal cells, much endotoxin is transported to and concentrated in the walls.     

Cytoplasmic pH-dependent spreading of polymorphonuclear leukocytes: regulation by pH of PKC subcellular distribution and F-actin assembly

Cytoplasmic pH (pHi) plays an important role in the regulation of polymorphonuclear leukocyte (PMN) spreading, but the molecular mechanisms involved have long been obscure. In the present study, we investigated the pH-dependence of phorbol myristate acetate (PMA)-induced PMN spreading. A change in pHi alone did not induce spreading, but cytoplasmic alkalinization promoted the spreading induced by PMA, whereas acidification inhibited it. To further investigate the mechanism by which pHi affects cell spreading, we employed subcellular fractionation and immunoblot analyses to evaluate the effect of pH on the subcellular distribution of protein kinase C (PKC) and assembly of the actin cytoskeleton. We found that cytoplasmic alkalinization enhanced PKC membrane distribution and quantitatively up-regulated the actin cytoskeleton. On the other hand, cytoplasmic acidification was found to have effects on these signaling molecules that were opposite to those of cytoplasmic alkalinization. These results may provide a potential explanation for the pH-regulation of the PMA-induced PMN spreading.