Monoclonal Antibody to CD14, TLR4, or CD11b: Impact of Epitope and Isotype Specificity on ROS Generation by Human Granulocytes and Monocytes

Lipopolysaccharides (LPSs or endotoxins) from Gram-negative bacteria represent pathogen-associated molecular patterns (PAMPs) that are recognized by CD14 and Toll-like receptor 4 (TLR4). Lipopolysaccharides prime polymorphonuclear leukocytes (PMNs) for substantial production of reactive oxygen species (ROS) during its response to secondary stimuli such as chemoattractants or pathogens. The excessive ROS production can damage surrounding host tissues, thereby amplifying the inflammatory reaction caused by pathogens. Today, specific antibodies against CD14, TLR4, and CD11b are being used as the essential tools to elucidate the role of these receptors in acute inflammation and some of these antibodies have advised as therapeutic agents for clinical use. Because each antibody has two antigen-binding arms [F(ab′)2] and one Fc arm, its effect on cellular response is much more complicated rather than simple blockage of target receptor. In fact, IgG antibody, once bound to target receptor, engages Fc receptors γ (FcγRs) and thereby is able to activate the adaptive immune system. The consequences of antibody-dependent binary heterotypic association of CD14, TLR4, or CD11b with FcγRs as well as homotypic one on ROS production are not well elucidated. Moreover, the consequences of antigenic recognition of CD14, TLR4, or CD11b by specific F(ab′)2 fragments are not always investigated. In this review, we will discuss known mechanisms underlying the therapeutic efficiency of CD14, TLR4, and CD11b/CD18 antibodies with a focus on LPS-dependent ROS or cytokine production by PMNs or monocytes. The impacts of F(ab′)2 as well as antibody IgG subclasses (isotypes) in therapeutic efficiency or agonistic potency of known antibodies against abovementioned receptors are presented. We also pay attention to how the efficiency of different IgG antibody subclasses is modulated during LPS-induced inflammation and by production of priming agents such as interferon γ (IFN-γ). Our review reinforces the molecular targets and therapeutic approaches to amelioration of harmful consequences of excessive activation of human pattern recognition receptors.


Introduction
Inflammatory and immune diseases affect millions of people worldwide, providing an impetus to develop new antiinflammatory and immunomodulatory therapies. Over the past two decades, great progress has been made in elucidating the molecular basis of the inflammation process during infec-tious, autoimmune, and malignant diseases [1][2][3][4]. It favors the development of new therapeutic drugs directly targeting cell surface or intracellular molecules involved in the initiation and progression of inflammation. In the case of endotoxemia, the major attention has been on application of LPS analogs with "under-acylated" lipid A structures, synthetic nontoxic lipid A derivatives, monoclonal antibodies to lipid A or truncated Re-LPS structure, blocking antibodies against both cell surface receptors and cytokines, and to other intracellular small molecule antagonists for therapeutic purposes. Today, a range of specific antibodies against CD14, TLR4, and CD11b are being used as the essential tool to elucidate their role in acute inflammation [1,[5][6][7][8][9][10][11][12][13][14][15][16]. We have shown earlier that some certain antibodies such as anti-CD11b ICRF-44 Fc mIgG1 or anti-TLR4 HTA125 Fc mIgG2a are unable to ameliorate significantly the N-formyl-methionylleucyl-phenylalanine-(fMLP-) triggered ROS production (luminol) from LPS-primed PMNs [12,13], while anti-CD14 UCHM-1 Fc mIgG2a suppresses LPS priming successfully [14]. Molecular mechanisms underlying our observations, however, have not been described. So, in this review, we discuss molecular mechanisms underlying LPS-induced functional responses of human PMNs and monocytes/macrophages such as ROS generation and production of cytokines after cell exposure to mouse IgG (mIgG) antibody to CD14, TLR4, or CD11b.
ROS is a collective term that often includes not only superoxide anion radical (O 2 ⋅− ) but other oxygen radicals such as hydroxyl (OH − ), peroxyl (RO 2 ), alkoxyl (RO ⋅ ), hydroperoxyl (HO 2 ⋅ ), and also nonradicals as hydrogen peroxide (H 2 O 2 ), hypochlorous acid (HOCl), singlet oxygen (ΔgO 2 ), and peroxynitrite (ONOO − ) [17]. Among them, H 2 O 2 is relatively stable diffusible oxidant acting as a signaling molecule and second messenger in the inflammatory settings. Today, it is known that signaling or damaging actions of ROS are determined by its amount, type, and cellular location. For example, H 2 O 2 has been shown to be involved in activation of nuclear factor NF-κB and probably in MAPK signaling cascades [18][19][20][21][22].
Circulating leukocytes are programmed for distinct functions in human physiology. The three main antimicrobial functions are accepted for PMNs: phagocytosis, degranulation, and the release of nuclear material in the form of PMN extracellular traps. Nowadays, it is recognized that PMNs can produce cytokines, modulate activities of neighboring cells, contribute to the resolution of inflammation, regulate macrophages for long-term immune responses, and even have a role in innate memory [23,24]. The main function of monocytes is the "processing" and degradation of antigens. Once produced from the bone marrow into the blood, circulating monocytes should be rapidly activated by inflammatory signals and migrate to areas of inflammation where they can differentiate into the proinflammatory (M1) or anti-inflammatory (M2) phenotypes known as tissues macrophages. In the M1 state, the activated monocytemacrophages undergo a metabolic switch from the oxidative phosphorylation to glycolysis. Inhibition of oxidative phosphorylation increases ROS production which exerts bactericidal activities. During the resolution of inflammation, abundance of anti-inflammatory (M2) monocytemacrophages with more oxidative phosphorylation phenotype is increased [25]. Classically activated M1 monocytemacrophages have elevated microbicidal function associated with the ability to secrete high amount of proinflammatory cytokines (TNF-α, IL-1β, and IL-12) and ROS, while alternatively activated M2 monocyte-macrophages produce high levels of anti-inflammatory mediators (IL-10 and TGF-β) [26].
ROS are by-products of numerous enzymatic reactions in various cell compartments, including the cytoplasm, cell membrane, endoplasmic reticulum, mitochondria, and peroxisome [18]. It has been suggested that peripheral blood monocytes depend on oxidative phosphorylation (ATP synthesis) for their energy supply, whereas PMNs do not. PMNs lose their mitochondrial dependency during maturation from bone marrow mitochondrion-rich precursors into peripheral blood PMNs with relative few mitochondria [30]. As the result, it has been proposed that mitochondria in PMNs (unlike monocyte-macrophages) do not play a role in energy metabolism, but maintain mitochondrial membrane potential for apoptotic signaling [25]. PMNs during phagocytosis use large quantities of molecular O 2 not for mitochondrial respiration, but, rather, to generate O 2 ⋅− and other oxidants via a respiratory burst catalyzed by NADPH oxidase [31].
NADPH oxidase is the enzyme responsible for O 2 ⋅− production [32]. This multicomponent enzyme system is composed of two transmembrane proteins (p22 phox and gp91 phox /NOX2 forming cytochrome b 558 ), three cytosolic proteins (p40 phox , p47 phox , and p67 phox ), and GTPase (Rac1 or Rac2). These components of NADPH oxidase are assembled at membrane sites upon transition of PMNs to a state of enhanced responsiveness known as priming. Three major events accompany activation of NADPH oxidase: (1) protein phosphorylation, (2) activation of GTPases, and (3) translocation of cytosolic components of NADPH oxidase to plasma membrane or to membrane of intracellular granules. Actually, NADPH oxidase in PMNs exists in different states: resting, primed, activated, or inactivated [33]. It has been demonstrated that O 2 ⋅− /ROS derived by NADPH oxidase are critically involved in LPS intracellular signaling leading to PMN priming as well as to maintenance of the resting or nonprimed state [34][35][36][37]. The primed PMNs have been identified in humans with infections, rheumatoid arthritis, chronic kidney disease, traumatic injury, and acute respiratory distress syndrome [38].
As known, PMNs express a range of receptors including β 2 integrins (CD11/CD18) and Fc receptors γ (FcγRs) which are capable of initiating complex intracellular signaling events robustly activating NADPH oxidase. In addition, some members of G protein-coupled receptors (GPCRs), especially fMLP receptor FPR1, can directly activate NADPH oxidase, although to a lesser extent than what has been 2 Oxidative Medicine and Cellular Longevity observed with activated integrins or FcγRs [32]. It is necessary to note that LPS itself does not elicit in PMN significant O 2 ⋅− /ROS production but transforms them into a primed state in which NADPH oxidase is not fully assembled but becomes more susceptible to activation by secondary stimuli [32-34, 39, 40].
An amplified O 2 ⋅− /ROS production from LPS-primed and fMLP-stimulated PMNs is the result at least of two converging intracellular signaling pathways. The first LPS-induced signaling pathway engages in PMNs such adaptor proteins as MyD88, TIRAP/MAL, IRAK, TRAF6, and TAK1. Among them, TAK1 is linked to MAPK signaling cascades [52]. After 20 minutes of PMN exposure to LPS, the MKK3-dependent phosphorylation of p38 MAPK is observed [53]. The p38 MAPK-dependent translocation of cytochrome b 558 and p47 phox but not p67 phox or Rac2 to the plasma membrane is also known. fMLP in LPS-primed PMNs causes a rapid and strong translocation of the other cytosolic components of NADPH oxidase to the already mobilized cytochrome b 558 followed by O 2 ⋅− /ROS production [54].

Heterotrimeric G iα2 Proteins and Their Intracellular Signaling Events
The second fMLP-initiated pathway is realized by FPR1 coupled with heterotrimeric G iα2 proteins. The activated G βγ subunit of G iα2 initiates concomitant activation of phospholipase C (PLC) and PI3K signaling pathways. The activity of p38 MAPK and ERK1/2 kinases is also upregulated during activation of G proteins [55][56][57]. Activated PLC hydrolyses phosphatidylinositol 4,5-bis-phosphate [PtdIns(4,5)P 2 or PI(4,5)P 2 ] in the plasma membrane leading to production of inositol 1,4,5-triphosphate [Ins(1,4,5)P 3 ] that is followed by Ca 2+ release from intracellular stores and generation of diacylglycerol (DAG), which in turn activates protein kinase C (PKC). The increase in intracellular free Ca 2+ leads to Ca 2+ influx into the cell. A rise in Ca 2+ is an essential step in PMN activation and O 2 ⋅− /ROS generation. Activated PKC induces phosphorylation of several substrates including p47 phox of NADPH oxidase. At the same time, activated PI3K produces phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P 3 or PI(3,4,5)P 3 ] from PtdIns(4,5)P 2 . The ability of wortmannin to inhibit PI3K and to abolish the fMLP-triggered respiratory burst without any effect on agonist-induced [Ca 2+ ] i flux or PKC-mediated NADPH oxidase activation has provided strong evidence to support a second-messenger role for PtdIns (3,4,5
In monocytes, LPS-induced release of proinflammatory cytokines is mediated by PI3K in both a ROS-and G protein-dependent manner, propagated through NADPH oxidase complex 4 (NOX4). Upregulation of PKB/Akt is completely inhibited by pretreatment of human PBMC with either pertussis toxin (inhibitor of G αi PCRs) or apocynin (inhibitor of NADPH oxidase 4) [21].

Human Fcγ Receptors and Their Ligands
Monoclonal antibodies to cell surface receptors such as CD14, TLR4, or CD11b/CD18 have various modes of actions. The simplest mode of their action is mere binding of the antibody to its antigen, thereby interfering or not with receptor activation. On the other hand, the antibody is able to block receptor interaction with their ligand, interfering with a multimerization process or triggering internalization of the receptor. In addition, once bound to antigen, IgG antibodies can engage the adaptive immune system via the interaction of their constant Fc region with FcγRs [59]. The human FcγR family contains six known members in three subgroups, including CD64, CD32 (CD32A, B, C), and CD16 (CD16A, B) [60]. CD32A is mainly expressed on monocytes (5 × 10 4 /cell), macrophages, and PMNs (1 -4 × 10 4 /cell), whereas CD32B is on B cells principally [11,[61][62][63]. The cytoplasmic domain of CD32A contains the immunoreceptor tyrosine-based activation motif (ITAM), while CD32B the immunoreceptor tyrosine-based inhibitory motif (ITIM) [11]. Human CD64 is abundantly expressed on monocytes (15 -40 × 10 3 /cell) while at lower levels on PMNs (1 -2 × 10 3 /cell) and macrophages (1 × 10 5 /cell) [61,62]. Human CD64 could be engaged by human IgG1 or mouse IgG2a (mIgG2a) but not mIgG1 or mIgG2b. Human CD32 appears to be engaged by mIgG1 or mIgG2b preferentially [64][65][66][67][68][69][70]. It is becoming increasingly evident that many receptors on myeloid cells do not act in isolation, but rather cooperate with other receptors to 3 Oxidative Medicine and Cellular Longevity coordinate responses to stimuli. For example, during immune complex (IC) recognition by the cell, CD11b/CD18 cooperates with CD16B (1 -3 × 10 5 /PMN) to cause Ca 2+ flux and ROS generation. CD16 and CD11b/CD18 jointly prime CD32 for ROS generation [71,72]. In human PMNs, both CD32 and CD16B are able to upregulate PI3K activity. Moreover, simultaneously engaged CD32 and CD16B via "insideout" signaling can recruit and activate CD11b/CD18 on PMNs. Thus, three different types of interaction between FcγRs and integrins could be realized: (1) a physical interaction on the cell surface, (2) integrin response that occurs because of FcγRs engagement and "inside-out" signalization, and (3) cellular responses to FcγRs that occur only after integrin occupation or when both receptors are stimulated simultaneously, i.e., "outside-in" signalization [73].
CD11b is able to regulate PtdIns(4,5)P 2 generation at the cell membrane through an ADP-ribosylation factor (ARF6)-PIP5K pathway. The increase in PtdIns(4,5)P 2 levels causes association of adaptor protein TIRAP/MAL with the plasma membrane, where it is needed to recruit MyD88 to TLR4 [74]. The functional coupling of aggregated CD64 to the PLD-and PKC-dependent activation of NADPH oxidase in IFN-γ-primed and IC-stimulated human monocytic U937 cells has been shown earlier. On the other hand, CD32A is coupled to PLC but is independent of PLD activation [75].

CD14-Associated Intracellular
Signaling Events CD14 is the most excessively studied TLR4 gatekeeper. Because CD14 is a glycosylphosphatidylinositol-(GPI-) anchored membrane protein without a transmembrane sequence, it is believed that CD14 has no intrinsic signaling ability during LPS recognition by innate immune cells. However, the LBP·LPS complex initially binds to CD14 and only then LPS is presented to the MD-2·TLR4 complex. CD14 controls the generation of PtdIns(4,5)P 2 that is required for maximal LPS-induced TLR4-dependent proinflammatory signaling [76]. Moreover, CD14 is essential for LPSdependent activation of phospholipases and MAPKs [77]. All together, these facts indicate CD14 as a promising therapeutic target. The impact of CD14 in TLR4-initiated signaling events has been studied in several works [64,[78][79][80][81][82] including our own [14].
Thus, in monocytes, Ca 2+ signaling could be induced by antibody-dependent association of CD14 with the high affinity receptor CD64. In addition, antibody-dependent homotypic CD32 association (CD32 ← anti-CD32 CIKM5 Fc mIgG1 → CD32) is also able to induce Ca 2+ mobilization. However, association of two CD32 is less effective for Ca 2+ mobilization when compared to heterotypic CD14 association with CD64. When CD64 is saturated, the lower affinity CD32A may also be engaged by mIgG2a antibody (CD14 ← anti-CD14 UCHM-1 Fc mIgG2a → CD64/CD32). It is necessary to note that anti-CD14 UCHM-1 Fc mIgG2a -induced Ca 2+ mobilization is weaker than that caused by fMLP [64]. Thus, G protein-coupled FPR1 appeared to be a more potent inductor of Ca 2+ signaling than the engagement of CD32 or CD64 (note not clustering). Unexpectedly, Ca 2+ mobilization in monocytes exposed to anti-CD14 UCHM-1 Fc mIgG2a antibody has been not associated with O 2 ⋅− generation (SOD-inhibitable ferricytochrome C reduction) [64]. Although in our settings the anti-CD14 UCHM-1 Fc mIgG2a antibody caused certain priming effect on fMLP-triggered O 2 ⋅− /ROS production by human PMNs, we did not observe any statistically significant differences [14]. The data from other works have suggested that anti-CD14 UCHM-1 Fc mIgG2a antibody is able to elicit in monocytes or PMN sufficient signal for phosphoinositide breakdown and Ca 2+ mobilization but it is not enough to initiate the assembly of NADPH oxidase and O 2 ⋅− /ROS generation [14,78]. Sufficient mobilization of Ca 2+ in all monocytes but only in subset of PMN (40%) has been detected only after CD14 crosslinking by anti-CD14 Mo2 Fc mIgM or anti-CD14 MEM-18/63D3 Fc mIgG1 antibodies followed by secondary F(ab ′ ) 2 fragments. The broad homotypic aggregation (crosslinking) of CD14 in the plane of plasma membrane has been suggested to be responsible for the robust increase in H 2 O 2 /ROS production in monocytes while less pronounced in PMNs [80,81]. The higher sensitivity of monocytes to antibody-dependent initiation of Ca 2+ signaling in comparison to PMNs can be explained by the differences in CD14 levels on their cell surfaces [84].
The Ca 2+ mobilization induced by CD14 crosslinking is suppressed when PLC or protein tyrosine kinases (PTK) have been inhibited [80]. Thus, only broad CD14 aggregation is able to stimulate substantial rise in intracellular free Ca 2+ and O 2 ⋅− /ROS production. CD14 in monocytes is physically associated with nonreceptor PTK Src Lyn p53/56 . The crosslinking of CD14 leads to Src Lyn p53/56 activation followed by concomitant upregulation of Src Frg p58 and Src Hck p59/61 kinases [85,86]. Earlier studies have shown that the signaling events triggered by CD14 crosslinking were abolished when the GPI anchor had been replaced by transmembrane sequence, suggesting that the localization to lipid rafts endowed CD14 with signaling ability [79,80]. As GPI-anchored receptors have high lateral mobility in the plane of cell membrane, they may be more easily aggregated upon interaction with a specific ligand [81]. Thus, CD14 would function to concentrate LPS at the cell surface for their recognition by other LPSbinding proteins and to facilitation of PtdIns(4,5)P 2 generation [87]. 4 Oxidative Medicine and Cellular Longevity

Epitope Specificity and Effectiveness of Anti-CD14 Antibodies against LPS-Induced Effects
LPS-binding sites on CD14 have been intensively studied, and four regions within the NH 2 -terminal 65 amino acid residues are identified. All of these regions (R) are clustered around the hydrophobic pocket of CD14. R1 (D 9 DED 12 ) is located close to the wall, whereas R3 (A 35 VEVE 39 ) is at the bottom of the pocket, while R2 (P 22 QPD 25 ) and R4 (D 57 ADPRQY 63 ) are located at the rim of the pocket. Another three regions of CD14, namely, T1 (E 7 LDDEDF 13 ), T2 (L 91 RVLAYSRLKE 101 ), and T3 (P 185 GL), have been proposed to be involved in LPS transfer to MD-2·TLR4 and therefore are responsible for LPS signaling. R1 within CD14 overlaps with T1 region. Therefore, the T1/R1 sequence appears to play a role in both LPS binding and transfer (LPS signaling) to MD-2·TLR4 [88,89]. The effectiveness of various anti-CD14 antibodies against LPS-induced effects is listed in Table 1.
6.1. Targeting R1 and T1 by 3C10 Antibodies Interferes with LBP·LPS Binding to CD14. Anti-CD14 3C10 Fc mIgG2b antibody binds to mostly anionic E 7 LDDEDFR 14 sequence in CD14 that is able to interact with cationic proteins such as serum LBP [106,111,112]. Anti-CD14 3C10 antibody almost completely prevents PMN priming by LBP·LPS for fMLP- /ROS production (luminol) [90]. So, the first antibody-dependent mechanism downregulating LPS deleterious effects is based on the ability of anti-CD14 antibodies to prevent the binding of LBP·LPS to CD14 and to abolish subsequent LPS transfer to MD-2·TLR4 [106,111].

Targeting R4 by MEM-18 Antibodies Suppresses LBP·LPS
Binding to CD14. Anti-CD14 MEM-18 Fc mIgG1 antibody binds to L 51 -A 64 sequence in R4 region (D 57 -A 64 ) of CD14. It is able to interfere with entry of lipid A, the hydrophobic region of LPS, into the hydrophobic pocket of CD14 (R4) during CD14 recognition of LBP·LPS, thereby suppressing the harmful effects caused by LPS [113][114][115][116]. Thus, despite the differences in isotype, the anti-CD14 MEM-18 Fc mIgG1 antibody, similar to anti-CD14 3C10 Fc mIgG2b antibody, prevents the binding of LPS to CD14 [113,117]. As a result, LPSinduced production of both TNF-α from human monocytes [97,118] or IL-8 from PBMC [119] has been suppressed. The effectiveness of anti-CD14 MEM-18 Fc mIgG1 antibody can be explained also by its supplementary ability to downregulate CD14 and TLR4, but not CD11b/CD18, from the cell surface as has been shown earlier using differentiated monocytic THP-1 cells [113,120]. Since anti-CD14 MEM-18 is a mIgG1 antibody and may be recognized by FcγRs (CD14 ← anti-CD14 MEM-18 Fc mIgG1 → CD64/CD32), its mode of action is more complicated [113,121].
It is necessary to note that LPS-induced IL-8 production has been shown to be suppressed more effectively by anti-CD14 MEM-18 antibody than anti-TLR4 HTA125 Fc mIgG2a antibody [119]. The effectiveness of anti-CD14 UCHM-1 Fc mIgG2a antibody against LPS-induced effects in human monocytes is less evident than that of anti-CD14 MEM-18 Fc mIgG1 antibody [97,118].
6.3. Targeting R3 by MY4 Antibodies Causes Internalization of CD14 and TLR4. Anti-CD14 MY4 Fc mIgG2b antibody binds to S 34 -G 44 sequence of CD14 and does not prime human PMNs for fMLP-triggered O 2 ⋅− /ROS production (reduction of ferricytochrome C) [104,113] but suppresses LBP·LPSinduced CD11b/CD18 mobilization to the cell surface [87]. Moreover, both LPS-induced association of G iα2 with PMN plasma membrane and activation of PLD are significantly suppressed by prior cell exposure to anti-CD14 MY4 antibody [104]. The effectiveness of anti-CD14 MY4 Fc mIgG2b antibody against LPS-induced effects is associated with its ability to induce downregulation of CD14 and TLR4 from the cell surface. It is interesting to note that LPS-independent internalization of CD14 and TLR4 during cell response to anti-CD14 MY4 Fc mIgG2b antibody exceeded that of anti-CD14 MEM-18 Fc mIgG1 antibody [100,104,113]. Thus, the effectiveness of anti-CD14 MY4 antibody against LPSinduced effects is based on its ability to block LBP·LPS binding to CD14 and to downregulate CD14 and TLR4 from the cell surface. Why mIgG2b antibodies to CD14 (MY4) or TLR4 (HT4) are internalized better than mIgG1 anti-CD14 MEM-18 antibody remain to be elucidated.

Anti-CD14 Antibodies as a Constituent of Therapeutic
Medications. CD14 as evidenced from data presented in Table 1 is involved in LPS-dependent PMN priming [103]. The relative weak effectiveness of anti-CD14 UCHM-1 Fc mIgG2a antibody as a suppressor of LPS-dependent PMN priming for fMLP-triggered O 2 ⋅− /ROS production may be explained by its epitope specificity that blocks CD14 incompletely [14]. However, the inhibitory effectiveness of anti-CD14 UCHM-1 antibodies may be improved by replacing their mIgG2a isotype with mouse or human IgG1.
The therapeutic relevance of anti-CD14 28C Fc mIgG1 or anti-CD14 18E12 Fc mIgG1 antibodies against LPS-induced effects has been already studied in vivo in INF-γ-sensitized Macaca fascicularis [109] and in normal human subjects (anti-CD14 IC14 Fc mhIgG1 ) [122]. Anti-CD14 28C/18E12 Fc mIgG1 antibodies protect primates from most of the physiologic and proinflammatory consequences of acute endotoxemia. The intravenous treatment of M. fascicularis by anti-CD14 18E12 antibody blocks signaling events without affecting the binding of LPS to CD14 as it has been estimated during LPS-induced production of TNF-α. On the other hand, productions of IL-6 and IL-1β have been inhibited better by another anti-CD14 28C antibody that is able to block LBP·LPS binding to CD14 [109]. A beneficial anti-CD14 IC14 Fc mhIgG1 antibody attenuates acute LPS-induced clinical symptoms and strongly inhibits LPS-induced production of proinflammatory cytokines, while it only delayed the release of the anti-inflammatory cytokines such as soluble TNF receptor type I and IL-1 receptor antagonist [122].
The extracellular region of TLR4 can be divided into Nterminal (L 52 -P 202 ), central (L 203 -L 348 ), and C-terminal /ROS production (luminol) [12,132]. The same result has been obtained by Sanui et al. [133]. These authors did not observe pronounced inhibitory effect of anti-TLR4 HTA125 antibody  [129] showed that the anti-TLR4 HTA125 antibody is able to suppress LPS-induced production of ROS (Phagoburst kit) in PMNs. When LPS-induced production of IL-6 and IL-8 was studied in the human embryonic cell line HEK293, no inhibitory effect of anti-TLR4 HTA125 Fc mIgG2a was detected [6,11]. However, in human retinal pigment epithelial cells, anti-TLR4 HTA125 antibody was almost equally as effective as anti-CD14 UCHM-1 Fc mIgG2a antibody in suppression of LPSinduced IL-8 production. It is interesting to note that simultaneous use of anti-TLR4 HTA125 and anti-CD14 UCHM-1 antibodies did not further potentiate antibody inhibitory effectiveness, suggesting that blockage of initial LPS binding to CD14 was highly effective and not further increased when TLR4 was also targeted [110].
The weak inhibitory effectiveness of anti-TLR4 HTA125 -Fc mIgG2a antibody could be explained by the epitope specificity. This antibody recognizes an antigenic epitope within D 50 -I 190 sequence and binds to TLR4 irrespective of the presence or absence of MD-2 [132]. Thus, it may be suggested that blockage of LRR2-LRR7 (HTA125) is not enough to prevent LPS·MD-2-induced TLR4 dimerization (Table 2).

Targeting the C-Terminal Domain of TLR4.
Anti-TLR4 HT4 Fc mIgG2b antibody recognizes the nonlinear epitope within the LRR13 repeat of TLR4. This epitope is composed of several amino acid residues (K 349 , K 351 S 352 , G 364 NA, and S 368 E) closely located to the TLR4 dimerization interface created by LRR15-LRR17 repeats of two LPS·MD-2·TLR4 complexes. Based on experimental data, it has been assumed that anti-TLR4 HT4 antibody is unable to prevent LPS·MD-2 binding to TLR4 but nevertheless inhibits LPS·MD-2induced TLR4 internalization. The effectiveness of anti-TLR4 HT4 Fc mIgG2b antibody in suppression of lipid Ainduced production of TNF-α, IL-6, and IL-12p40 from human leukocytes is better than that of the anti-TLR4 HTA125 Fc mIgG2a antibody [1,5,6]. Taking these facts into consideration, it could be concluded that targeting the C-terminal domain of TLR4 may lead to a more pronounced therapeutic effect than targeting the N-terminal domain of TLR4 by HTA125 mIgG2a or HT52 mIgG1 antibodies. The effect of anti-TLR4 HT52 Fc mIgG1 (LRR2-LRR7: D 50 -I 190 sequence) antibody may be potentiated by simultaneous application with anti-TLR4 HT4 Fc mIgG2b (LRR13) antibody thus causing double blocking of TLR4 (LRR2-LRR7 and LRR13) [1,5,6]. Note that anti-TLR4 HTA125 antibody recognizes the same antigenic epitope as did anti-TLR4 HT52 antibody. We assume that double targeting TLR4 by anti-TLR4 HT4 and anti-TLR4 HTA125 antibodies would improve the inhibitory effectiveness of the latter.
As has been shown experimentally, the inhibitory effectiveness of anti-TLR4 HT4 Fc mIgG2b or anti-TLR4 HT52 Fc mIgG1 is not associated with engagement of FcγRs. In fact, the inhibitory effectiveness of anti-TLR4 HT4 Fc mIgG2b or anti-TLR4 HT52 Fc mIgG1 against lipid A-induced effects was unaffected by prior cell exposure to blocking anti-CD32 AT-10 Fc mIgG1 antibody [1, 5, 6].

Targeting Both N-and C-Terminal Domains of TLR4.
The improved antibody effectiveness seen with doubletargeted TLR4 led to the generation of a new anti-TLR4 15C1 Fc mIgG1 antibody recognizing both LRR12 (Y 328 N) and LRR13 (K 349 LK, E 369 VD) sequences [11]. Anti-TLR4 15C1 antibody blocks TLR4 binding to LPS·MD-2 and TLR4 dimerization as well. In addition, anti-TLR4 15C1 antibody effectively suppresses LPS-induced IL-6 and IL-8 production analogous to anti-TLR4 HT4 Fc mIgG2b or anti-TLR4 HT52 Fc mIgG1 antibodies but with stronger effect than anti-TLR4 HTA125 Fc mIgG2a antibody [6,11]. Furthermore, anti-TLR4 15C1 antibody prevents LPS-induced TLR4 partitioning into lipid rafts [61]. As earlier has been shown, the effectiveness of anti-TLR4 15C1 Fc mIgG1 antibody is dependent on the engagement of FcγRs (CD32) [1,11]. Targeting CD32 by anti-CD32 IV.3/AT-10 Fc mIgG2b antibodies dramatically reduces the effectiveness of anti-TLR4 15C1 Fc mIgG1 antibody when LPS-induced production of IL-6 was studied. It is necessary to note that LPS-induced IL-6 production had not been significantly affected by isotype-matched control mIgG1. Thus, in addition to engagement of CD32 (TLR4 ← anti-TLR4 15C1 Fc mIgG1 → CD32) and its signaling pathway(s), the therapeutic effect of anti-TLR4 15C1 Fc mIgG1 antibody is based on its ability to prevent LPS·MD-2 binding to TLR4 thereby abolishing TLR4 dimerization and its movement into lipid rafts [1,11,134].

Humanized Anti-TLR4 Antibody and FcγRs.
The differences in the affinity of FcγRs for IgG subclasses have been explored in development of new therapeutic antibodies such as Hu15C1. This antibody is the humanized version of anti-TLR4 15C1 Fc mIgG1 antibody [61,135]. Two substitutions (N 325 → S and L 328 → F) have been introduced into Fc arm of anti-TLR4 15C1 Fc mIgG1 antibody to amplify its inhibitory effectiveness. As a result, the affinity of the new anti-TLR4 Hu15C1 Fc hIgG1 for CD64 is potentiated, while for CD16, it is eliminated. An intermediate affinity of anti-TLR4 Hu15C1 Fc hIgG1 for CD32 was detected. Thus, CD64 (TLR4 ← anti-TLR4 Hu15C1 Fc hIgG1 → CD64/CD32) is viewed as the first contributor to the potent inhibitory effectiveness of anti-TLR4 Hu15C1 Fc hIgG1 antibody. In addition, CD32initiated ITAMi signaling is expected when CD64 would be not available. The blockage of CD32B does not change significantly the inhibitory effectiveness of anti-TLR4 Hu15C1 antibody. When anti-TLR4 Hu15C1 antibody had been compared with the parental anti-TLR4 15C1 Fc mIgG1 antibody, the former antibody was more effective than the latter in inhibition of LPS-induced effects. In addition, it has been shown using neuronal originated HEK293 cells that CD32A is involved in the inhibitory potency of anti-TLR4 Hu15C1 antibody. It was also discovered that the inhibitory potency of anti-TLR4 Hu15C1 antibody against LPS-induced effects is much higher in CD32A-positive HEK293 cells than in CD32A-negative ones. Besides engagement of CD64 and CD32, the anti-TLR4 Hu15C1 antibody is able to interfere with LPS-induced TLR4 dimerization thereby preventing TLR4 partitioning into lipid rafts. Thus, it is postulated that dimerization of TLR4 is a prerequisite for TLR4 clustering [61]. 7 Oxidative Medicine and Cellular Longevity

) Suppress LPS-induced internalization of TLR4
(2) Probably stimulate dimerization of two TLR4 (1) Interfere with transfer of mCD14·LPS to MD-2·TLR4 and LPS-induced TLR4 dimerization as well [6,131] HTA405 (isotype not pointed out) (1) Suppress production of TNF-α and IL-8 from THP-1/CD14 + cells stimulated by Re-LPS S. minnesota R595 (10 ng/ml) The therapeutic effectiveness of humanized anti-TLR4 NI-0101 antibody has been already evaluated ex vivo and in vivo in the absence or presence of systemic LPS challenge (acute inflammation) [7,9]. The other effective neutralizing anti-TLR4 WN1222-5 antibody mimics the site of TLR4, recognizing an inner core structure of LPS from most infectious bacteria regardless of the presence of Opolysaccharide (O-antigen) and prevents LPS binding to target cells in the bloodstream [136]. 7.5. Impact of CD32 in Effectiveness of IgG Antibodies. Antigenic recognition of CD32A by anti-CD32 IV.3 Fab/F(ab′) 2 fragments recognizing the F 132 SHLDP 137 sequence does not cause Ca 2+ mobilization, upregulation of p38 and ERK MAPK kinases, and intracellular production of H 2 O 2 /ROS (scopoletin) [137]. The other anti-CD32 CIKM5 Fab/F(ab ′ ) 2 are also unable to induce Ca 2+ mobilization when crosslinking by secondary F(ab ′ ) 2 fragments is omitted [64,78]. Targeting CD32 by anti-CD32 AT-10 F(ab′) 2 inhibits constitutive and fMLP-triggered O 2 ⋅− /ROS (luminol) production by PMNs [134,138]. In PMNs, a small rise in intracellular free Ca 2+ has been induced also by anti-CD32 CIKM5 Fc mIgG1 antibody but it was not associated with O 2 ⋅− release (SOD-inhibitable reduction of ferricytochrome C) [64,78]. Antigenic recognition of CD32A by anti-CD32 AT-10 Fab fragments recognizing an epitope located near or within IgG-binding site fails to activate differentiated monocytic THP-1 cells [134,138]. Recent findings however have indicated that antigenic CD32 recognition by anti-CD32 IV.3/AT-10 Fab/F(ab ′ ) 2 induced ITAMi signaling [134]. It has been shown that engagement of CD32 by anti-CD32 AT-10 F(ab ′ ) 2 stimulates transient recruitment of tyrosine kinase 2SH2 Syk/p72 Tyr to cytoplasmic domain of CD32A followed by incomplete phosphorylation of the ITAM leading to the inhibitory ITAMi conformation. As a result, activated ITAMi allows tyrosine phosphorylation of SHP-1 (protein tyrosine phosphatase) followed by inhibition of the major intracellular signaling players of immune cells such as guanine nucleotide exchange factor Vav-1 (RacGEF) and IRAK-1 kinase that are both involved in O 2 ⋅− /ROS and cytokine production. Thus, blockage of Vav-1 in human PMNs can abrogate association of p67 phox with NADPH oxidase thereby suppressing O 2 ⋅ − /ROS production [134]. This suppressive effect of CD32A and mIgG1 might be reversed by LPS-induced assembly and stabilization of TLR4, CD11b/CD18, and FcγRs in lipid rafts followed by the activation of classical ITAM signaling [11,41,44,79,139]. Since PMNs express very low levels of CD32B, an impact of its involvement in the inhibitory effectiveness of mIgG1 antibodies should be negligible [140].
In humans, as it has been revealed in vivo, the LPS administration causes an initial rapid decline in the absolute PMN counts at 1 h followed by an increase that reaches a maximum at 6 h and then declines at 22 h to basal levels. In addition, LPS injection induces biphasic CD64 upregulation on circulating PMNs. The first phase has been observed after 1 h of LPS challenge, while the second started at 6 h and reached a maximum at 22 h [148].
By contrast, freshly isolated monocytes constitutively express high levels of surface CD64 [61,62] and respond to anti-CD11b VIM12 Fc mIgG1 or anti-CD11b Leu-15 Fc mIgG2a antibodies by intracellular ROS production (DCFDA). For example, anti-CD11b Leu-15 Fc mIgG2a antibody causes an increase in ROS production in monocytes of all responders, while agonistic anti-CD11b VIM12 Fc mIgG1 in 60% only. When anti-CD11b VIM12 Fc mIgG1 antibody has been devoid of the Fc arms, its ability to stimulate ROS (DCFDA) production in freshly isolated monocytes was diminished [63]. Thus, the agonistic activity of anti-CD11b VIM12 Fc mIgG1 antibody represents the cumulative effect of epitope specificity and capability to engage CD64/CD32 receptors.
The engagement of CD64, and CD32 to a lesser extent, by anti-TLR4 HTA125 Fc mIgG2a antibody has been already revealed using macrophage-like Mono Mac 6 cells (CD32 > CD64). Mouse IgG2a inhibits most effectively the binding of anti-TLR4 HTA125 Fc mIgG2a antibody to Mono Mac 6 cells, while mIgG1 or mIgG2b reveals no significant effect. Further, the binding of anti-TLR4 HTA125 antibody to Mono Mac 6 cells has been most effectively prevented by anti-CD64 10.1 Fc mIgG1 antibody by contrast to anti-CD32 FLI8.26 Fc mIgG2b antibody. Targeting CD16 by anti-CD16 3G8 Fc mIgG1 antibody does not influence significantly on anti-TLR4 HTA125 binding to Mono Mac 6 cells [62]. Based on these findings, the high affinity of human CD64 and moderate affinity of human CD32 for the constant Fc arm of mIgG2a antibodies can be concluded [62,63]. These results indicate also that specific Fab or F(ab ′ ) 2 fragments against cell surface receptors would be most appropriate and safe for applying in "antibody"-based therapy.

CD11b/CD18 and Their Signaling Partners
Macrophage-1 antigen (Mac-1, α M β 2 , or CD11b/CD18) is a complement receptor (CR3). It consists of noncovalently linked CD11b (integrin α M ) and CD18 (integrin β 2 ) subunits. Integrins regulate important leukocyte functions including adhesion, migration, proteolysis, phagocytosis, and oxidative (respiratory) burst [150]. In resting PMNs, integrins are maintained in a conformationally inactive state and are unable to bind their ligands [151]. On cell stimulation, "inside-out" signaling originating from nonintegrin cell surface receptors such as FPR1, FcγRs, or TLR4 leads to vast conformational changes in CD11b/CD18, but not directly to receptor clustering (integrin redistribution in the plane of plasma membrane) [42,52]. Thus, only "inside-out" primed integrins exhibit increased ligand-binding avidity and initiate "outside-in" signaling by themselves. CD11b/CD18 has been detected in the LPS-induced "receptor cluster" on monocytes and can act as a signaling partner for such receptors as FPR1 and CD14 [44,106,150,152,153]. CD11b/CD18 can be found also in association with FcγRs, but the consequences of these functional interactions are not fully understood. The functional association of the GPI-anchored form of CD16 ( PMN CD16B GPI ) with CD11b/CD18 is mediated by the lectin-binding site of the latter [147,151,[153][154][155]. The mechanisms by which CD11b/CD18 regulates leukocyte functions such as respiratory burst are still poorly understood.
Like other β 2 integrins, CD11b consists of a short cytoplasmic tail, single transmembrane domain, and long extracellular domain. The extracellular domain of CD11b is composed of seven repeats. The V to VII repeats are similar to the divalent cation-binding "EF-hand" motif. The II and III repeats are separated by the I (inserted) domain that is known also as αA/I-domain [156]. The effectiveness of anti-CD11b/CD18 antibodies against LPS-induced effects is listed in Table 3.
8.1. Targeting the CD11b αA/I-Domain. Antigenic recognition of CD11b/CD18 by anti-CD11b 44 Fab recognizing αA/I-domain does not lead to considerable changes in conformation of CD11b/CD18 nor to activation of "outside-in" signaling [161,162]. However, full anti-CD11b 44a or anti-CD11b/CD18 IB4 Fc mIgG2a antibodies induce epitope exposition by CD11b that is recognized by anti-CD11b VIM12 Fc-mIgG1 antibody [147]. This effect may be explained by antibody-dependent association of CD11b/CD18 with FcγRs. So, 44a or IB4 antibodies are able to induce "outside-in" signaling followed by "inside-out" signaling leading to further conformational changes in CD11b/CD18.  [137]. In PMNs, neither anti-CD11b 60.1/44 Fc mIgG1 nor anti-CD11b/CD18 IB4 Fc mIgG2a antibodies cause Ca 2+ mobilization [70,145,146]. The same result was obtained during cell response to anti-CD18 MHM23 Fc mIgG1 or anti-CD11b/CD18 60.3 Fc mIgG2a antibodies regardless of isotype differences [78,145]. Further, intracellular H 2 O 2 /ROS (DHR) triggered in PMNs by antibody-dependent association of CD32 with CD11b/CD18 (CD11b/CD18 ← anti-CD11b 44/IB4 Fc mIgG1/mIgG2a ) was less pronounced than that in response to agonistic anti-CD11b VIM12 Fc mIgG1 antibody [147]. Thus, it can be concluded that simple antibody-dependent association of CD11b/CD18 with CD32 (regardless of epitope specificity) is not enough to initiate significant agonistic (activating) signaling events and H 2 O 2 /ROS production in PMNs, whereas to induce sufficient intracellular signaling by IgG1 antibodies, targeting of a particular epitope on CD11b is required. In fact, when agonistic anti-CD11b VIM12 Fc mIgG1 antibody recognizing the CD11b lectin site had been employed to PMNs, "outside-in" signaling, clustering of activated CD11b/CD18, upregulation of PI3K and PKB/Akt signaling pathways, Ca 2+ mobilization, and actin polymerization were all realized, but the Raf → ME-K1/2 → ERK1/2 signaling pathway was not upregulated [163]. Agonistic anti-CD11b VIM12 Fc mIgG1 antibody prevents association of CD11b/CD18 with CD16B GPI [163] but nevertheless induces PMNs for generation of H 2 O 2 /ROS (DHR) to the same extent as fMLP. Anti-CD11b VIM12 Fc mIgG1 -dependent production of H 2 O 2 /ROS (DHR) exceeds that induced by IB4 or 44a antibodies [147]. Taking these facts into consideration, it may be suggested that CD11b/CD18-dependent "outside-in" signaling would be successfully realized when CD11b lectin domain is docked (thus, CD11b/CD18 is primed) by other appropriate GPI-anchored protein on the same cell. In PMNs, "outside-in" signaling is also initiated by CD11b/CD18 that has been clustered in the response to anti-CD11b 2LPM19c F(ab′) 2 recognizing αA/I-domain. The same effect had been observed when whole anti-CD11b 2LPM19c Fc mIgG1 antibody was used. Unexpectedly, monovalent anti-CD11b 2LPM19c Fab is unable to produce such agonistic activity [158]. These results clearly show that agonistic activity of anti-CD11b antibodies is determined by F(ab ′ ) 2 epitope specificity. Moreover, F(ab′) 2 can potentially bind two targets leading to close proximity of two integrin molecules that mimic receptor crosslinking [164].
In PMNs, generation of H 2 O 2 /ROS (DHR) induced by anti-CD11b VIM12 Fc mIgG1 antibody is not diminished by prior cell exposure to anti-CD11b 44a Fc mIgG1 or anti-CD11b/CD18 IB4 Fc mIgG2a antibodies. It necessary to note that sequential treatment of PMNs in whole blood by anti-CD11b/CD18 IB4 Fc mIgG2a and anti-CD11b VIM12 Fc mIgG1 antibodies led to more pronounced generation of H 2 O 2 /ROS (DHR) than treatment with anti-CD11b 44a Fc mIgG1 combined with anti-CD11b VIM12 Fc mIgG1 antibody [147]. This result may be explained by the fact that anti-CD11b/CD18 IB4 Fc-mIgG2a antibody is able to potentiate the binding of anti-CD11b VIM12 Fc mIgG1 antibody to CD11b/CD18 in addition to their ability to affect both subunits of CD11b/CD18. Thus, anti-CD11b/CD18 IB4 Fc mIgG2a antibody, besides binding to CD11b/CD18, can also engage other β 2 integrins via the common CD18 subunits (CD11a/CD18 and CD11c/CD18). Such broad targeting of β 2 integrins could provide sufficient 11 Oxidative Medicine and Cellular Longevity signals for intracellular H 2 O 2 /ROS generation (DHR). This conclusion is supported by the fact that antigenic recognition of CD11b/CD18 by anti-CD11b/CD18 IB4 F(ab ′ ) 2 induces Ca 2+ mobilization to a similar extent as whole anti-CD11b/CD18 IB4 Fc mIgG2a antibody [165].
In PMNs, generation of ROS/H 2 O 2 (DHR) induced by anti-CD11b VIM12 Fc mIgG1 antibody can be blocked almost completely by prior cell exposure to anti-CD32 IV.3 Fc mIgG2b antibody [160]. Thus, unlike monocytes, CD32 receptor on PMNs is involved in CD11b/CD18-induced generation of ROS/H 2 O 2 (DHR) in the response to anti-CD11b VIM12 Fc mIgG1 antibody (CD11b/CD18 ← anti-CD11b VIM12 Fc mIgG1 → CD32). Therefore, it has been supposed that association of CD11b/CD18 with CD32 is required for ROS generation during PMN response to anti-CD11b VIM12 Fc mIgG1 antibody [147]. Ortiz-Stern and Rosales [73] have shown that CD32 and CD11b/CD18 are uniformly distributed and not colocalized on the surface of unstimulated PMNs. A similar observation has been made for PMN CD16B GPI which is also uniformly distributed across the cell surface and is not colocalized with CD32 in unstimulated PMNs [73].
By contrast to PMNs, in monocytes, a close spatial proximity between CD11b/CD18 and CD32/CD64 has been suggested by Gadd et al. [63]. These authors have observed that anti-CD11b VIM12 Fc mIgG1 antibody interferes sterically with anti-CD32 IV.2 Fc mIgG2b antibody for binding to CD32 on monocytes, but not on PMNs [63]. Interestingly, in their study, anti-CD11b VIM12 Fc mIgG1 antibody had been unable to induce intracellular ROS (DCFDA) generation in PMNs as they did in monocytes [63,147].

Anti-CD11b/CD18 Antibodies in Therapeutic
Medications. In the light of data presented here, the effectiveness of anti-CD11b/CD18 antibodies against LPS-induced effects should be discussed. It has been shown that CD11b/CD18 has a site recognizing LPS carbohydrates, namely, N-acetyl-D-glucosamine (GlcNAc) and mannose. In addition, CD11b/CD18 binds truncated LPS glycoforms such as Re-LPS [153][154][155]. Two putative LPS-binding sites within the CD18 βA region (AA 216-248 , 266-318 ) had been proposed earlier [166]. It was found that Re-LPS from Salmonella minnesota is bound through cationic interactions by the βA 266-318 -exposed CD18 sequence, while another βA 216-248 sequence probably utilizes other interactions like hydrophobic ones [166]. Thus, it is not excluded that 3-deoxy-Dmanno-octulosonic acid (KDO), the inner core sugar of almost all LPS molecules, may be involved in LPS recognition by CD18 [153,155,159,160]. However, neither LPS binding to PBMC nor LPS-induced PMN priming for fMLPtriggered ROS production has been blocked by anti-CD11b OKM1 Fc mIgG1 antibody [91,99,100,104]. A similar result has been obtained in our work [13] where we used anti-CD11b ICRF44 Fc mIgG1 antibody. This antibody binds probably to αA/I-domain of CD11b (AA 201-217 , 245-261 ) in a manner independent of inactive or active CD11b/CD18 state [158,162,167]. Thus, it may be concluded that targeting the αA/I-domain of CD11b by anti-CD11b ICRF44 Fc mIgG1 or the CD11b lectin site by anti-CD11b OKM1 Fc mIgG1 antibodies is unable to suppress significantly LPS-dependent PMN prim-ing for fMLP-triggered O 2 ⋅− /ROS production [13,91,99,100,104]. From the data presented here, we can, however, not exclude that other CD11b/CD18 sites might be involved in LPS recognition. The effectiveness of anti-CD11b 44 Fc mIgG1 antibodies as suppressors of LBP·LPS-dependent PMN priming for fMLP-triggered O 2 ⋅− /ROS production is less pronounced than that of anti-CD14 3C10 Fc mIgG2b antibody [90]. Thus, targeting CD14 by appropriate antibodies would be more effective in comparison to targeting CD11b/CD18 or TLR4. Moreover, it has been shown that CD11b/CD18 interacts more avidly with aggregated but not monomeric LPS and this interaction occurs even better in the absence of LBP [106,168]. Taking these data into consideration, we concluded that CD11b/CD18 is not an appropriate target for "antibody"-based therapy even when anti-CD11b Fab/F(ab ′ ) 2 fragments are used.

Conclusions
In summary, several conclusions can be drawn. Neither mIgG2a nor mIgG1 are able to stimulate Ca 2+ mobilization and ROS production in freshly isolated PMNs.
Sufficient signals for phosphoinositide breakdown could be induced in monocytes by CD14 association with CD64. Moreover, this heterotypic CD14 association with CD64/CD32 (anti-CD14 UCHM-1 Fc mIgG2a ) or association of two CD32 receptors together (CD32 ← anti-CD32 CIKM5 Fc-mIgG1 → CD32) leads to Ca 2+ signaling in monocytes. The homotypic CD32 association is less effective in Ca 2+ signaling than heterotypic association of CD14 with CD64. In spite of this, CD14 association with CD64 is not enough to trigger O 2 ⋅ − /ROS production in monocytes. Ca 2+ signaling caused by CD64/CD32 engagement without crosslinking is weaker than that induced by fMLP.
In monocytes not only sufficient production of PI(4,5)P 2 but also robust upregulation of intracellular H 2 O 2 /ROS is initiated in response to broad CD14 crosslinking by anti-CD14 mIgG1 antibodies and secondary F(ab ′ ) 2 . In PMNs, this effect of CD14 crosslinking is less pronounced. TLR4 crosslinking [anti-TLR4 76B351.1 Fc mIgG2b plus F(ab ′ ) 2 ] does not have the same effect on PI(4,5)P 2 production as CD14 crosslinking. Thus, PI(4,5)P 2 generation is a specific response to CD14 crosslinking (clustering).
Antigenic recognition of CD32 by anti-CD32 IV.3/AT-10 Fab/F(ab′) 2  Oxidative Medicine and Cellular Longevity nonagonistic targeting CD11b followed by its association with CD32 is not enough to activate PMNs for significant H 2 O 2 /ROS generation but it takes place when CD11b will be targeted by agonistic antibody followed by CD32 engagement. A high sensitivity of CD11b/CD18 to environmental stimuli including antibodies and its ability to initiate "inside-out" signaling makes CD11b/CD18 not an appropriate target for "antibody"-based therapy even if anti-CD11b Fab/F(ab′) 2 fragments would be used. By contrast to CD11b/CD18, targeting CD14 by Fab or F(ab′) 2 fragments is the most appropriate and safe approach for "antibody"-based therapy for LPS-induced deleterious effects even when innate immune cells are primed by PAMPs or endogenous priming molecules. A similar conclusion can be made accordingly targeting TLR4 by specific Fab/F(ab′) 2 fragments. The effectiveness of anti-TLR4 Fab/F(ab′) 2 fragments further may be potentiated by simultaneous use with anti-CD14 Fab/F(ab′) 2 . However, it is necessary to kept in mind that partial TLR4 blockage may cause the better therapeutic effect since certain TLR4 activation is required for development of the adaptive immune responses. Anti-TLR4 antibody is able to interfere with TLR4 binding to LPS·MD-2 thereby preventing TLR4 dimerization in addition to engagement of CD32 and CD64 signaling pathways.
In light of the data presented and based on our own observations [12,13,14], we can conclude that only anti-CD14 UCHM-1 Fc mIgG2a but neither anti-TLR4 HTA125 Fc mIgG2a nor anti-CD11b ICRF44 Fc mIgG1 antibodies are able to prevent significantly LPS-induced PMN priming for fMLP-triggered O 2 ⋅− /ROS generation. Therefore, we confirm that CD14 is really the main TLR4 gatekeeper. We believe that anti-CD14 Fab/F(ab′) 2 fragments will be very suitable for clinical use and could improve outcomes during LPS-initiated inflammation.